U.S. patent number 11,324,742 [Application Number 16/936,975] was granted by the patent office on 2022-05-10 for treatment of demyelinating diseases.
This patent grant is currently assigned to University of Kansas, Victoria Link Ltd.. The grantee listed for this patent is University of Kansas, Victoria Link Ltd.. Invention is credited to Bronwyn Maree Kivell, Anne Camille La Flamme, Thomas Edward Prisinzano.
United States Patent |
11,324,742 |
Prisinzano , et al. |
May 10, 2022 |
Treatment of demyelinating diseases
Abstract
The present invention relates generally to methods of using
nalfurafine for treating and/or preventing demyelinating disease in
a subject, and in particular for treating and/or preventing
multiple sclerosis (MS). Also disclosed is nalfurafine for use in
treating and/or preventing MS as well as pharmaceutical
compositions and unit dosage forms comprising nalfurafine for use
for treating and/or preventing demyelinating disease in a subject,
and in particular for treating and/or preventing MS.
Inventors: |
Prisinzano; Thomas Edward
(Lawrence, KS), Kivell; Bronwyn Maree (Wellington,
NZ), La Flamme; Anne Camille (Wellington,
NZ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Victoria Link Ltd.
University of Kansas |
Wellington
Lawrence |
N/A
KS |
NZ
US |
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Assignee: |
Victoria Link Ltd. (Wellington,
NZ)
University of Kansas (Lawrence, KS)
|
Family
ID: |
1000006292971 |
Appl.
No.: |
16/936,975 |
Filed: |
July 23, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210069179 A1 |
Mar 11, 2021 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/IB2019/051870 |
Mar 7, 2019 |
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Foreign Application Priority Data
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Mar 8, 2018 [AU] |
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2018900754 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
31/485 (20130101); A61P 25/28 (20180101) |
Current International
Class: |
A61K
31/485 (20060101); A61P 25/28 (20060101) |
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|
Primary Examiner: Cruz; Kathrien A
Attorney, Agent or Firm: Fish & Richardson P.C.
Government Interests
1. U.S. GOVERNMENT RIGHTS
This invention was made with government support under DA018151
awarded by the National Institutes of Health. The government has
certain rights in the invention.
Parent Case Text
CLAIM OF PRIORITY
This application is a continuation of PCT Application No.
PCT/IB2019/051870, filed on Mar. 7, 2019, which claims priority to
Australian Patent Application Serial No. 2018900754, filed on Mar.
8, 2018, the entire contents of both applications are hereby
incorporated by reference.
Claims
The claims defining the invention are as follows:
1. A method of treating multiple sclerosis (MS) in a human subject
in need thereof, the method comprising identifying a human subject
as having MS; and administering to the human subject daily, for a
period of at least 7 days, a pharmaceutical composition comprising
a therapeutically effective amount of about 2.5 .mu.g to about 83.0
.mu.g of nalfurafine, wherein the therapeutically effective amount
is sufficient to increase remyelination in the human subject,
wherein the increase in remyelination comprises increasing a
percentage of myelination area in the human subject's brain to a
level that is up to about 89% of a percentage of myelination area
in a healthy human brain.
2. The method of claim 1, wherein the daily dose of nalfurafine is
10.0 .mu.g nalfurafine.
3. The method of claim 1, wherein the method comprises daily
administration of the pharmaceutical composition for at least 14
days.
4. The method of claim 1, wherein the method comprises
administration of the pharmaceutical composition for at least one
month.
5. The method of claim 1, further comprising one or more of the
following steps selected from the group consisting of: diagnosing
MS in the subject; testing for remyelination in the subject;
testing for a level of paralysis in the subject; testing
coordination in the subject; and testing balance in the
subject.
6. A method of increasing remyelination in a human subject having
multiple sclerosis (MS), the method comprising identifying a
subject as having MS; and administering to the subject for a period
of at least 7 days a pharmaceutical composition comprising an
amount of nalfurafine effective to increase a level of
remyelination in the subject relative to a level of myelination in
the subject before administering the pharmaceutical composition,
wherein the amount of nalfurafine in the pharmaceutical composition
comprises about 0.1 to about 10.0 .mu.g nalfurafine.
7. The method of claim 6, wherein the method results in one or more
clinical outcomes selected from the group consisting of: a decrease
in MS disease progression; a decrease in MS disease severity; a
decrease or delay in nerve cell demyelination; a decrease in
frequency or severity of relapsing MS attacks; a healing of damaged
nerve tissue; an increase in remyelination of demyelinated nerves
in the subject's central nervous system; protection of damaged
nerve tissue from further disease activity; promotion of neuronal
outgrowth in the subject's central nervous system; an improvement
in nerve function; and an enhanced rate of remission.
8. The method claim 6, wherein the method results in a reduction of
one or more clinical symptoms of MS selected from the group
consisting of: loss of sensitivity, muscle weakness, impaired
walking, impaired hand function, pronounced reflexes, muscle
spasms, difficulty in moving, ataxia, spasticity, problems with
speech or swallowing, visual problems, fatigue, acute or chronic
pain, facial pain, incontinence, reduced cognitive ability,
depression, anxiety, sexual dysfunction, Uhthoff's phenomenon, and
Lhermitte's sign.
9. The method of claim 6, wherein the method comprises daily
administration of the pharmaceutical composition for at least 14
days.
10. The method of claim 6, wherein the method comprises
administration of the pharmaceutical composition for at least one
month.
11. The method of claim 6, further comprising one or more of the
following steps selected from the group consisting of: diagnosing
MS in the subject; testing for demyelination in the subject;
testing for a reduction in demyelination in the subject; testing
for remyelination in the subject; testing for a level of paralysis
in the subject; testing coordination in the subject; and testing
balance in the subject.
12. A method of attenuating demyelination in a subject having
multiple sclerosis (MS), the method comprising identifying a
subject as having MS; and administering to the subject for a period
of at least 7 days a pharmaceutical composition comprising an
amount of nalfurafine effective to decrease a level of
demyelination relative to a level of demyelination before
administering the pharmaceutical composition, wherein the amount of
nalfurafine in the pharmaceutical composition comprises about 0.1
to about 10.0 .mu.g nalfurafine.
13. The method of claim 12, wherein the method results in one or
more clinical outcomes selected from the group consisting of: a
decrease in MS disease progression; a decrease in MS disease
severity; a decrease or delay in nerve cell demyelination; a
decrease in frequency or severity of relapsing MS attacks; a
healing of damaged nerve tissue; an increase in remyelination of
demyelinated nerves in the subject's central nervous system;
protection of damaged nerve tissue from further disease activity;
promotion of neuronal outgrowth in the subject's central nervous
system; an improvement in nerve function; and an enhanced rate of
remission.
14. The method claim 12, wherein the method results in a reduction
of one or more clinical symptoms of MS selected from the group
consisting of: loss of sensitivity, muscle weakness, impaired
walking, impaired hand function, pronounced reflexes, muscle
spasms, difficulty in moving, ataxia, spasticity, problems with
speech or swallowing, visual problems, fatigue, acute or chronic
pain, facial pain, incontinence, reduced cognitive ability,
depression, anxiety, sexual dysfunction, Uhthoff's phenomenon, and
Lhermitte's sign.
15. The method of claim 12, wherein the method comprises daily
administration of the pharmaceutical composition for at least 14
days.
16. A method of reducing one or more clinical symptoms of multiple
sclerosis (MS) in a human subject having MS, the method comprising
identifying a subject as having MS; and administering to the
subject for a period of at least 7 days a pharmaceutical
composition comprising an amount of nalfurafine effective to
decrease a clinical symptom in the subject relative to a level of
the clinical symptom in the subject before administering the
pharmaceutical composition, wherein the amount of nalfurafine in
the pharmaceutical composition comprises about 0.1 to about 10.0
.mu.g nalfurafine.
17. The method of claim 16, wherein the method further results in
one or more clinical outcomes selected from the group consisting
of: a decrease in MS disease progression; a decrease in MS disease
severity; a decrease or delay in nerve cell demyelination; a
decrease in frequency or severity of relapsing MS attacks; a
healing of damaged nerve tissue; an increase in remyelination of
demyelinated nerves in the subject's central nervous system;
protection of damaged nerve tissue from further disease activity;
promotion of neuronal outgrowth in the subject's central nervous
system; an improvement in nerve function; and an enhanced rate of
remission.
18. The method claim 16, wherein the method results in a reduction
of one or more clinical symptoms of MS selected from the group
consisting of: loss of sensitivity, muscle weakness, impaired
walking, impaired hand function, pronounced reflexes, muscle
spasms, difficulty in moving, ataxia, spasticity, problems with
speech or swallowing, visual problems, fatigue, acute or chronic
pain, facial pain, incontinence, reduced cognitive ability,
depression, anxiety, sexual dysfunction, Uhthoff's phenomenon, and
Lhermitte's sign.
19. The method of claim 16, wherein the method comprises daily
administration of the pharmaceutical composition for at least 14
days.
20. The method of claim 1, wherein the daily dose of nalfurafine is
about 8.3 .mu.g to 83.0 .mu.g of nalfurafine.
21. The method of claim 1, wherein the daily dose of nalfurafine is
about 2.5 .mu.g to 25 .mu.g.
Description
2. TECHNICAL FIELD
The disclosure relates generally to the use of nalfurafine (NalF)
in the prevention and treatment of demyelinating diseases, in
particular, multiple sclerosis.
3. BACKGROUND
The myelin sheath covers important nerve fibres in the central and
peripheral nervous system of mammals, helping to facilitate
transmission of neural impulses. Diseases that affect myelin
interrupt these nerve transmissions. The developing myelin sheath
can be affected by congenital metabolic disorders such as
phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Hurler's
syndrome, and Krabbe's disease. Demyelination can also occur in
adults as a result of injury, metabolic disorders, immune attack,
ischemia and toxic agents.
Demyelination impairs conduction of signals to the affected nerves,
causing deficiency of sensation, movement, cognition and other
functions. Demyelination of the central nervous system is
associated with multiple sclerosis (MS), Devic's disease, acute
disseminated encephalomyelitis, adrenoleukodystrophy,
leukoencephalopathy and Leber's optic atrophy. Demyelination of the
peripheral nervous symptom gives rise to diseases such as
Guillain-Barre syndrome, chronic inflammatory demyelinating
polyneuropathy, Charcot Marie Tooth (CMT) disease and progressing
inflammatory neuropathy.
Multiple sclerosis (MS) is the most well-known demyelination
disease, affecting about 2.5 million people worldwide. Sufferers
endure a range of symptoms including fatigue, vision problems,
numbness, cognitive impairment, incontinence, poor balance and
muscle weakness, ultimately leading to paralysis. MS can follow
four major disease courses, each of which can be mild, moderate or
severe: 1. Relapsing-Remitting MS (RRMS)--clearly defined attacks
(flare-ups) of worsening neurological function followed by partial
or complete remission 2. Primary-Progressive MS (PPMS)--slowly
worsening neurological function at variable rates, with no distinct
remission 3. Secondary-Progressing MS (SPMS)--an initial period of
RRMS is followed by a steady worsening, with or without flare-ups
and remissions 4. Progressive-Relapsing MS (PRMS)--steadily
worsening neurological function with clear flare-ups and partial or
no remission.
While there is no cure for MS, many FDA approved drugs such as
beta-interferon and glatiramer acetate are used to reduce relapse
rates and the formation of new lesions. Unfortunately, current
treatments are not very successful in preventing the disability
associated with MS and are more successful in treating RRMS than
other types. For example, current drugs are unable to stop or
reverse disease progression and disability. Clearly, alternative
treatments for MS are needed.
It is therefore an object of the present invention to go at least
some way towards meeting this need in the art, to provide products
and methods useful in the treatment of the disability associated
with MS and/or that are able to stop and/or reverse MS disease
progression and disability and/or to at least to provide the public
with a useful choice.
4. SUMMARY OF THE INVENTION
In one aspect the invention provides a pharmaceutical composition
comprising nalfurafine and pharmaceutically acceptable excipients
for treating a demyelinating disease in a subject in need
thereof.
In one aspect the invention provides a pharmaceutical composition
comprising nalfurafine and at least one pharmaceutically acceptable
excipient for use for treating a demyelinating disease in a subject
in need thereof.
In another aspect the invention provides unit dosage forms
comprising about 0.01 to about 5 mg of nalfurafine and at least one
pharmaceutically acceptable carrier or excipient. In one
embodiment, the unit dosage form comprises 0.05 to about 2.0 mg of
nalfurafine and at least one pharmaceutically acceptable carrier or
excipient. In one embodiment the unit dosage form comprises about
0.15 to about 0.6 mg nalfurafine and at least one pharmaceutically
acceptable carrier or excipient.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject comprising identifying a subject
who would benefit from a decreased level of demyelination and
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
In another aspect the invention provides a method of increasing
remyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
In another aspect the invention provides a method of increasing
remyelination in a subject comprising identifying a subject who
would benefit from an increased level of remyelination and
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
In another aspect the invention provides a method of increasing
remyelination in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for treating a demyelinating disease in a subject
in need thereof.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for increasing remyelination in a subject in need
thereof.
The invention also provides nalfurafine for use for treating a
demyelinating disease.
The invention also provides nalfurafine for use for increasing
remyelination.
In one embodiment the disease is a demyelinating myelinoclastic
disease.
In one embodiment the disease is a demyelinating leukodystrophic
disease.
In one embodiment the demyelinating disease is a central nervous
system demyelinating disease. In one embodiment the central nervous
system demyelinating disease is selected from the group comprising
MS (including clinically isolated syndrome; CIS), optic neuritis,
Devic's disease, inflammatory demyelinating diseases, central
nervous system neuropathies, myelopathies like Tabes dorsalis,
leukoencephalopathies, leukodystrophies, or a combination
thereof.
In one embodiment the demyelinating disease is MS.
In another embodiment the demyelinating disease is a peripheral
nervous system demyelinating disease. In one embodiment the
peripheral nervous system demyelinating disease is elected from the
group comprising Guillain-Barre syndrome and its chronic
counterpart, chronic inflammatory demyelinating polyneuropathy,
anti-myelin associated glycoprotein (MAG) peripheral neuropathy,
Charcot Marie Tooth (CMT) disease, copper deficiency and
progressive inflammatory neuropathy.
In another aspect the invention provides a method of attenuating
demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject and thereby attenuating a level of demyelination in the
subject relative to the level of demyelination when nalfurafine is
not administered.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for attenuating demyelination in a subject in need
thereof. In one embodiment, the subject is a human with MS.
The invention also provides nalfurafine for use for attenuating
demyelination in a subject in need thereof.
In another aspect the invention provides a method of treating MS in
a subject in need thereof, comprising administering a
therapeutically effective amount of nalfurafine to the subject.
In another aspect the invention provides a method of treating MS in
a subject in need thereof, comprising administering to the subject
a therapeutically effective amount of an agent that decreases a
level of demyelination in the subject relative to the level before
administering the agent and/or that increases a level of
remyelination in the subject in the subject relative to the level
before administering the agent, wherein the agent comprises
nalfurafine.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for treating MS in a subject in need thereof.
The invention also provides nalfurafine for use for treating MS in
a subject in need thereof.
In one embodiment the subject has RRMS. In one embodiment the
subject has PPMS. In one embodiment the subject has, or is
diagnosed as having, SPMS. In one embodiment the subject has, or is
diagnosed as having, PRMS. In one embodiment the subject has, or is
diagnosed as having, Clinically Isolated Syndrome (CIS).
In one embodiment the treatment of MS results in one or more
clinical outcomes when compared to subjects not treated with
nalfurafine selected from the group consisting of: (a) a decrease
in MS disease progression; (b) a decrease in MS disease severity;
(c) a decrease in nerve cell demyelination; (d) a decrease in
frequency or severity of relapsing MS attacks; (e) a decrease in MS
clinical symptoms; (f) the healing of damaged nerve tissue
(neuro-restoration); (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); (h) the protection of damaged nerve
tissue from further disease activity (neuroprotection); (i) the
promotion of neuronal outgrowth (neuro-regeneration) in the central
nervous system; (j) a decrease in disability caused by MS; (k) an
improvement of nerve function; and (l) an enhanced rate of
remission.
In another embodiment the treatment of MS results in a reduction of
one or more clinical symptoms of MS including, but not limited to
loss of sensitivity or changes in sensation such as tingling, pins
and needles or numbness, muscle weakness or paralysis of variable
severity, very pronounced reflexes, muscle spasms, or difficulty in
moving; difficulties with coordination and balance (ataxia);
spasticity; problems with speech or swallowing, visual problems
(nystagmus, optic neuritis or double vision), fatigue, acute or
chronic pain, neuropathic pain, facial pain (trigeminal neuralgia),
bladder and bowel difficulties, incontinence, reduced cognitive
ability, depression, anxiety and other emotional abnormalities,
sexual dysfunction, Uhthoff's phenomenon (a worsening of symptoms
due to exposure to higher than usual temperatures), and Lhermitte's
sign (an electrical sensation that runs down the back when bending
the neck).
In one aspect the invention provides a method of accelerating
remission from MS in a subject in need thereof, the method
comprising administering a therapeutically effective amount of
nalfurafine to the subject.
In one aspect the invention provides a method of accelerating
remission from MS in a subject in need thereof, the method
comprising administering a therapeutically effective amount of an
agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
In one aspect the invention provides a method of accelerating
remission from MS in a subject in need thereof, the method
comprising administering a therapeutically effective amount of an
agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for accelerating remission from MS in a subject in
need thereof.
The invention also provides nalfurafine for use for accelerating
remission from MS in a subject in need thereof.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject comprising identifying a subject
who would benefit from a decreased level of demyelination and
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination relative to the
level of demyelination before administering the agent, wherein the
agent comprises nalfurafine.
In another aspect the invention provides a method of increasing
remyelination in a subject comprising identifying a subject who
would benefit from an increased level of remyelination and
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination relative to the
level of remyelination before administering the agent, wherein the
agent comprises nalfurafine.
In the above methods of the invention:
In one embodiment the therapeutically effective amount for a
subject is equivalent to a dose of about 0.003 to about 0.3
mg/kg/day in mice.
In one embodiment the subject is human. In one embodiment the
method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine, preferably less than 1 ug nalfurafine
daily.
In some embodiments the method comprises a long duration
therapy.
In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5 days, at least 6 days,
or at least 7 days.
In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least a week, at least 2 weeks,
at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 8
weeks.
In some embodiments the long duration therapy comprises
administration for at least 5 days, at least 6 days, at least 7
days, at least 14 days, at least 21 days, at least 28 days, at
least 35 days, at least 42 days, at least 45 days, at least 60
days, at least 120 days, at least 240 days, or at least 360
days.
In some embodiments the long duration therapy comprises a dosing
gap of at least 1 day.
Other aspects of the invention may become apparent from the
following description which is given by way of example only and
with reference to the accompanying figures.
In this specification where reference has been made to patent
specifications, other external documents, or other sources of
information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art. However, these
external documents and references are all cited herein by reference
in their entireties or at least to the extent described herein.
It is intended that reference to a range of numbers disclosed
herein (for example, 1 to 10) also incorporates reference to all
rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9,
4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational
numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1
to 4.7) and, therefore, all sub-ranges of all ranges expressly
disclosed herein are hereby expressly disclosed. These are only
examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
Whenever a range is given in the specification, for example, a
temperature range, a time range, or a composition range, all
intermediate ranges and subranges, as well as all individual values
included in the ranges given are intended to be included in the
disclosure. In the disclosure and the claims, "and/or" means
additionally or alternatively. Moreover, any use of a term in the
singular also encompasses plural forms.
5. BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described by way of example only and with
reference to the drawings in which:
FIG. 1 is a graph showing the progression of disease in mice which
have experimental autoimmune encephalomyelitis (EAE) over 45 days,
wherein the mice in Example 1 were treated with 0.01, 0.03, 0.1 or
0.3 mg/kg nalfurafine daily from onset (day 17).
FIGS. 2A-B are two graphs showing the total disability of EAE mice
over (A) 45 days and (B) 18 days wherein the mice in Example 2 were
treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from onset
(day 17).
FIG. 3 is a graph showing the % weight change of EAE mice in
Example 3 over 45 days wherein the mice were treated with 0.03, 0.1
or 0.3 mg/kg nalfurafine daily from onset (day 17).
FIGS. 4A-C are three graphs showing immune cell infiltration into
the brain of EAE mice in Example 4 after 45 days, wherein the mice
were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from
onset (day 17).
FIG. 5 is a graph showing the progression of disease in EAE mice in
Example 5 over 45 days, wherein the mice, which had not yet
developed EAE, were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine
daily from onset (day 17).
FIGS. 6A-C are a series of Transmission Electron Microscope (TEM)
images of spinal cord sections from EAE mice in Example 6 after 45
days, wherein the mice were treated with 0.03 mg/kg nalfurafine
daily from onset (day 17).
FIG. 7 is a graph showing weight gain over 65 days of mice in
Example 7 treated with 0.3% cuprizone for 5 weeks, wherein the mice
were treated with 0.1 mg/kg nalfurafine daily from week 4.
FIG. 8 is a graph showing the rotarod performance score of mice in
Example 8 at 9 weeks treated with cuprizone for 5 weeks, wherein
the mice were treated with 0.1 mg/kg nalfurafine daily from week
4.
FIGS. 9A-D are a series of TEM imagines of the corpus callosum of
mice in Example 9 at 9 weeks treated with cuprizone for 5 weeks,
wherein the mice were treated with 0.1 mg/kg nalfurafine daily from
week 4.
FIG. 10 shows that nalfurafine promotes functional recovery from
paralysis when administered therapeutically (at disease onset) in
the experimental autoimmune encephalomyelitis (EAE) model of
MS.
FIG. 11 shows that nalfurafine is not effective when administered
therapeutically as a short 4-day course starting at disease onset
in EAE model of MS.
FIG. 12 shows that nalfurafine does not alter peak disease when
administered therapeutically in the EAE model of MS.
FIG. 13 shows that nalfurafine promotes full recovery from
EAE-induced paralysis when administered therapeutically.
FIG. 14 shows that nalfurafine promotes full recovery from
EAE-induced paralysis when administered therapeutically with an
EC50 for % recovery of <0.001 mg/kg.
FIG. 15 shows that nalfurafine promotes sustained recovery from
EAE-induced paralysis when administered therapeutically.
FIG. 16 shows that nalfurafine also promotes functional recovery
from paralysis in male mice when administered therapeutically in
EAE model of MS.
FIG. 17 shows that nalfurafine also promotes full recovery in male
mice when administered therapeutically in EAE model of MS.
FIG. 18 shows that nalfurafine promotes sustained recovery in male
mice from EAE-induced paralysis when administered
therapeutically.
FIGS. 19A-B show that nalfurafine reduces the immune cell
infiltration into the brain when administered therapeutically in
the EAE model of MS (A) whereas U 50488 does not (B).
FIG. 20 shows that myelination is improved in mice treated with
nalfurafine after the onset of paralysis in the EAE model of
MS.
FIGS. 21A-C show that nalfurafine does not alter the proportion of
major lymphocyte populations in the spleen during the chronic phase
of EAE.
FIGS. 22A-D show that nalfurafine does not alter the overall number
of CD4 T helper cells in the spleen but shifts the CD4 T cells from
an effector to memory phenotype being suggestive of immune
resolution during the chronic phase of EAE.
FIG. 23 shows that nalfurafine reduces disease but does not enable
full recovery when the kappa opioid receptor (KOR) is blocked.
FIGS. 24A-C show that activation of the KOR is required for full
recovery from paralysis mediated by nalfurafine.
FIGS. 25A-D show that myelination is improved in mice treated with
nalfurafine after the onset of paralysis in the EAE model of
MS.
FIGS. 26A-C show that nalfurafine treatment decreases cellular
infiltration into the spinal cord when administered therapeutically
in the EAE model of MS.
FIGS. 27A-1, 27A-2, and 27B show that nalfurafine treatment reduces
the level of activated astrocytes in the spinal cord when
administered therapeutically in the EAE model of MS.
FIGS. 28A-C show that nalfurafine treatment enhances recovery from
weight loss when administered therapeutically in the cuprizone
model of MS.
FIGS. 29A-G show that nalfurafine treatment enhances remyelination
in the brain when administered after demyelination in the cuprizone
demyelination disease model of MS.
FIGS. 30A-B show that nalfurafine is more effective at promoting
functional recovery than clemastine fumarate, a known remyelinating
drug.
FIGS. 31A-1, 31A-2, 31B-1, and 31B-2 show that nalfurafine promotes
a greater and more sustained recovery than clemastine fumarate, a
known remyelinating drug.
FIGS. 32A-1, 32A-2, and 32B show that nalfurafine promotes recovery
in pain threshold when administered after demyelination in the
cuprizone demyelination disease model of MS.
6. DETAILED DESCRIPTION
6.1 Nalfurafine
Nalfurafine is a drug commonly prescribed for treatment of uremic
pruritus in people with chronic kidney disease. It is a
non-narcotic opioid with selective K-opioid receptor (KOR) agonist
activity. The inventors have now found that nalfurafine is a
surprisingly effective treatment for demyelinating diseases.
The generic name "nalfurafine" refers to the compound:
##STR00001##
The IUPAC name for nalfurafine is
(E)-N-[(4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-1,2,4,5,-
6,7,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-yl]-3-(fura-
n-3-yl)-N-methylprop-2-enamide. Its CAS number is 152657-84-6.
Nalfurafine HCl may also be referred to as
17-cyclopropylmethyl-3,14-beta-dihydroxy-4,5-alpha-epoxy-6beta-(N-methyl--
trans-3-(3-furyl)acrylamido)morphinan hydrochloride, TRK 820,
AC-820 and MT-9938.
As used herein the term "nalfurafine" refers to the compound
identified above as well as to its pharmaceutically acceptable
salts and solvates.
The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids
including inorganic or organic bases and inorganic or organic
acids. Salts derived from inorganic bases include aluminum,
ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic salts, manganous, potassium, sodium, zinc, and the like.
Particularly preferred are the ammonium, calcium, magnesium,
potassium, and sodium salts. Salts in the solid form may exist in
more than one crystal structure and may also be in the form of
hydrates. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylene-diamine, diethylamine, 2-diethylaminoethanol,
2-dimethylamino-ethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like. When nalfurafine is
basic, salts can be prepared from pharmaceutically acceptable
non-toxic acids, including inorganic and organic acids. Such acids
include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,
ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric, isethionic, lactic, maleic, malic, mandelic,
ethanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,
succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
Particularly preferred are citric, hydrobromic, hydrochloric,
maleic, phosphoric, sulfuric, fumaric, and tartaric acids.
The term "solvate" refers to an aggregate that consists of a solute
ion or molecule with one or more solvent molecules. "Solvates"
include hydrates, that is, aggregates of a compound of interest
with water.
Nalfurafine can be purchased from small molecule suppliers such as
Med Chem Express, Monmouth Junction and New Jersey, USA; AdooQ
BioScience, Irvine Calif., USA.
6.2 Pharmaceutical Compositions of Nalfurafine
There is a lack of effective treatments for demyelinating diseases,
including MS, and in particular, there are few effective agents
that act to reduce demyelination and/or to increase remyelination.
Surprisingly, the inventors have found that pharmaceutical
compositions containing nalfurafine can be used to treat
demyelination diseases including but not limited to MS by acting to
increase remyelination and/or to decrease demyelination.
Accordingly, in one aspect the invention provides a pharmaceutical
composition comprising nalfurafine and pharmaceutically acceptable
excipients for treating a demyelinating disease in a subject in
need thereof.
In another aspect the invention provides a pharmaceutical
composition comprising nalfurafine and at least one
pharmaceutically acceptable excipient for use for treating a
demyelinating disease in a subject in need thereof.
This term "pharmaceutical composition" as used herein encompasses a
product comprising one or more active agents, and pharmaceutically
acceptable excipients comprising inert ingredients, as well as any
product which results, directly or indirectly, from combination,
complexation or aggregation of any two or more of the ingredients,
or from dissociation of one or more of the ingredients, or from
other types of reactions or interactions of one or more of the
ingredients. In general, pharmaceutical compositions are prepared
by bringing the active agent into association with a liquid
carrier, a finely divided solid carrier or both, and then, if
necessary, shaping the product into the desired formulation. Said
compositions are prepared according to conventional mixing,
granulating, or coating methods, respectively, and contain a
percentage (%) of the active ingredient and can be determined by a
skilled worker in view of the art.
The term "comprising" as used herein means "consisting at least in
part of". When interpreting each statement in this specification
that includes the term "comprising", features other than that or
those prefaced by the term may also be present. Related terms such
as "comprise" and "comprises" are to be interpreted in the same
manner.
The term "consisting essentially of" as used herein means the
specified materials or steps and those that do not materially
affect the basic and novel characteristic(s) of the claimed
invention.
The term "consisting of" as used herein means the specified
materials or steps of the claimed invention, excluding any element,
step, or ingredient not specified in the claim.
By "pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" it is meant that the excipient or carrier must
be compatible with the other ingredients of the formulation and not
harmful to the subject to whom the composition is administered.
Pharmaceutical compositions as described herein can be administered
topically, orally or parenterally.
For example, the pharmaceutical compositions can be administered
orally, including sublingually, in the form of capsules, tablets,
elixirs, solutions, suspensions, or boluses formulated to dissolve
in, for example, the colon or duodenum. The formulations can
comprise excipients such as starch or lactose or flavouring,
preserving or colouring agents.
The pharmaceutical compositions can be injected parenterally, for
example, intravenously, intramuscularly or subcutaneously. For
parenteral administration, the compositions can be formulated in a
sterile aqueous solution or suspension that optionally comprises
other substances, such as salt or glucose.
The compositions can be administered topically, in the form of
sterile creams, gels, pour-on or spot-on formulations, suspensions,
lotions, ointments, dusting powders, drug-incorporated dressings,
shampoos, collars or transdermal patches. For example, the
compositions as described herein can be incorporated into a cream
comprising an aqueous or oily emulsion of polyethylene glycols or
liquid paraffin; an ointment comprising a white wax soft paraffin
base; a hydrogel with cellulose or polyacrylate derivatives or
other suitable viscosity modifiers; a dry powder; aerosol with
butane, propane, HFA, or CFC propellants; a dressing, such as, a
tulle dressing, with white soft paraffin or polyethylene glycol
impregnated gauze dressings or with hydrogel, hydrocolloid, or
alginate film dressings. The compositions can also be administered
intra-ocularly as an eye drop with appropriate buffers, viscosity
modifiers (for example, cellulose derivatives), and preservatives
(for example, benzalkonium chloride).
The pharmaceutical compositions as described herein can also be
incorporated into a transdermal patch comprising nalfurafine.
Details of such patches can be found in, for example,
WO2015/025766, WO2015/025767, WO2016/208729, WO2017/094337 and
WO2017/170933, the details of which are incorporated by reference
herein.
For oral administration, capsules, boluses, or tablets can be
prepared by mixing the pharmaceutical compositions as described
herein with a suitable finely divided diluent or carrier,
additionally containing a disintegrating agent and/or binder such
as starch, lactose, talc, or magnesium stearate.
For parenteral administration injectable formulations can be
prepared in the form of a sterile solution or emulsion.
The compositions described herein can be presented in unit dosage
form and can be prepared by any of the methods well known in the
art of pharmacy. The term "unit dosage form" means a single dose
wherein all active and inactive ingredients are combined in a
suitable system, such that the patient or person administering the
drug can open a single container or package with the entire dose
contained therein and does not have to mix any components together
from two or more containers or packages. Typical examples of unit
dosage forms are tablets or capsules for oral administration or
transdermal patches comprising the unit dosage. These examples of
unit dosage forms are not intended to be limiting in any way, but
merely to represent typical examples in the pharmacy arts of unit
dosage forms.
In another aspect the invention provides unit dosage forms
comprising about 0.01 to about 5 mg of nalfurafine and at least one
pharmaceutically acceptable carrier or excipient. In one
embodiment, the unit dosage form comprises 0.05 to about 2.0 mg of
nalfurafine and at least one pharmaceutically acceptable carrier or
excipient. In one embodiment the unit dosage form comprises about
0.15 to about 0.6 mg nalfurafine and at least one pharmaceutically
acceptable carrier or excipient.
In one aspect the invention provides a unit dosage form comprising
about 0.1 to about 10 .mu.g of nalfurafine and at least one
pharmaceutically acceptable carrier or excipient. In one embodiment
the unit dosage form comprises about 0.5 to about 7.5 .mu.g
nalfurafine, about 0.75 to about 5 .mu.g nalfurafine, about 1 to 4
.mu.g nalfurafine, about 2-3 .mu.g nalfurafine, about 2 .mu.g
nalfurafine, about 3 .mu.g nalfurafine, about 4 .mu.g nalfurafine
or about 5 .mu.g nalfurafine.
In one embodiment the unit dosage form comprises less than about 2
.mu.g, 1.5 .mu.g, 1.0 .mu.g, 0.5 .mu.g, 0.25 .mu.g or 0.1 .mu.g,
preferably less than 2 .mu.g, 1.5 .mu.g, 1.0 .mu.g, 0.5 .mu.g, 0.25
.mu.g or 0.1 .mu.g.
In another embodiment, the unit dosage form is for treating a
demyelinating disease in a subject in need thereof, preferably
wherein the subject has MS. In another embodiment, the unit dosage
is formulated for treating a demyelinating disease in a subject in
need thereof. In one embodiment the demyelinating disease is
MS.
In another embodiment the unit dose is formulated for increasing
remyelination in a subject in need thereof, preferably wherein the
subject has MS.
In one embodiment, the unit dosage form is for oral administration,
preferably the unit dosage form is formulated for oral
administration. In another embodiment, the unit dosage form is a
transdermal patch.
The term "about" as used herein means a reasonable amount of
deviation of the modified term such that the end result is not
significantly changed. For example, when applied to a value, the
term should be construed as including a deviation of +/-5% of the
value.
Pharmaceutical compositions of nalfurafine can be used in
combination with other therapies for treating demyelination
diseases.
6.3 Therapeutic Uses of Nalfurafine
The inventors have surprisingly found that nalfurafine gives rise
to many positive effects in demyelination in MS mouse models. For
example, the inventors have found that nalfurafine is effective at
treating demyelination in mouse models of EAE and cuprizone-induced
demyelination, results that are translatable to treating
demyelinating diseases such as MS in humans. The inventors have
also found that nalfurafine is unexpectedly effective at increasing
remyelination in subjects in need thereof. Accordingly, this drug,
which has a proven safety record, could be highly beneficial in the
treatment of demyelination diseases and/or for increasing
remyelination.
As set out in Examples 1, 10 and 12-18, nalfurafine promotes
functional (including full and sustained) recovery from EAE-induced
paralysis in male and female mice. Nalfurafine also reduces
EAE-induced total disability (see Example 2) and promotes recovery
from EAE-induced weight loss (see Example 3). Importantly, the
disease score is reduced completely in the examples described
herein to <0.5, which is considered to represent a "full
recovery" from paralysis in the art, with one exception. A short
4-day time course starting at disease outset was not effective at
promoting recovery (Example 11), demonstrating the efficacy of a
long duration therapy as described herein.
Nalfurafine reduces immune cell infiltration into the brain in the
EAE model of MS (see Example 4) and is more effective than the
comparator U-50488, which does not (Example 19. When administered
before onset, nalfurafine promotes functional recovery from
paralysis, in the EAE model of MS (see Example 5). Myelination is
also improved in mice treated with nalfurafine after the onset of
paralysis in the EAE model of MS (Examples 6, 20 and 25).
By the examples described herein the inventors show clearly that
nalfurafine induces and/or increases remyelination in the EAE
model. In Example 6, TEM images of the spinal cords of EAE mice
treated with nalfurafine resemble those of the healthy control.
The EAE results were confirmed by cuprizone studies described in
Examples 7-9 and 11. In Examples 7 and 28, nalfurafine improved
weight gain when administered after cuprizone-induced
demyelination. In Example 8, nalfurafine enhanced the functional
recovery of coordination and balance in demyelinated mice.
Remyelination of the corpus callosum occurred when
cuprizone-treated mice were administered nalfurafine (see Examples
9 and 29).
In Example 15 the demonstration of sustained recovery is noteworthy
and shows the quite unexpected ability of nalfurafine to reverse,
in a sustained manner, the symptoms of demyelination. This
surprising result indicates that nalfurafine can mediate sustained
recovery of demyelinating diseases including MS.
In Example 21, nalfurafine does not deplete the major immune cell
populations in the periphery despite reducing immune cell
infiltration into the brain. In example 22, nalfurafine promotes a
switch in T helper cells from effector to memory cells suggestive
of immune response resolution.
In Examples 23 and 24, the KOR is required for the full effect of
nalfurafine but nalfurafine is effective at reducing disease
independently of the KOR suggesting the full mechanism by which
nalfurafine exerts its effects is more complex than KOR
activation.
The positive effects of nalfurafine on mice were particularly
surprising at dosages of 0.003 mg/kg to 0.3 mg/kg, which can be
converted to an equivalent human dose using the Regan-Shaw equation
(Reagan-Shaw S; Nihal M; Ahmad N: Dose translation from animal to
human studies revisited, FASEB J. 2007, Oct. 17).
Alternatively, dosages of 0.003 to 0.3 mg/kg can be converted to an
equivalent human dose using the method of interspecies comparison
described herein.
The skilled worker in the art appreciates that there are
alternative algorithms that can be used to convert an observed
therapeutic dosage from a mouse model into an equivalent human dose
once the effective mouse dosage has been demonstrated. Such
algorithms can be used effectively by the skilled person to
determine the appropriate human dose
For example, using a method of interspecies comparison, a skilled
worker employs the ratio of the efficacy dose for itch vs the
efficacy dose for MS in the same species. This ratio can be applied
to the human dose to convert dosage for itch to the dosage for MS.
In this case, there is dose data for treating itch in both mouse
and human models, and this enables the calculations described
below.
61/968,897 Data describing the drug dose that produces 50% of the
maximal effect (ED.sub.50).
TABLE-US-00001 Complete Route of ED.sub.50 Inhibition Mouse Model
Administration (.mu.g/kg) (.mu.g/kg) Reference Substance P IV 3.77
7.5-10 Winfuran-Assessment induced report European Medicines
scratch Agency, Committee for Substance P SC 1.65 10 Medicinal
Products for induced Human Use scratch (EMA/CHMP/138212/2014)
Substance P PO 9.61 100 induced (66%) scratch Morphine SC 2.34 5-10
induced scratch Histamine PO 7.3 30-100 Togashi et al. (2002).
induced Antipruritic activity of the scratch .kappa.-opioid
receptor agonist, Substance P PO 19.6 100 TRK-820. Eur 3 Pharmacol
induced 435:259 scratch
For itch model the average in vivo efficacy ED.sub.50 is
.about.2.71 .mu.g/kg (rounded up to 3 .mu.g/kg) by SC or IV
administration (only the data in the top two rows of the table
above were used in this calculation). The rationale for this is:
Administration in our EAE study was intraperitoneal (i.p.)
Bioavailability of nalfurafine (as described in
Winfuran--Assessment report European Medicines Agency, Committee
for Medicinal Products for Human Use (EMA/CHMP/138212/2014): oral
(PO) administration is .about.32% subcutaneous (s.c.) is 96%
intravenous (IV) is 100% Therefore, s.c. and IV administration will
have a similar bioavailability to i.p., whereas PO administration
will not due to first-pass effect of hepatic metabolism, and
therefore it has been excluded from the calculations. Additionally,
the morphine induced scratch model works through a mechanism of
action unrelated to that of substance P itch, and therefore was
excluded. Converting Dosage for Itch vs EAE
The calculation assumes that itch response is a biomarker
(surrogate) for EAE. 1. Mouse dose for itch is 3 .mu.g/kg/day 2.
Mouse dose for EAE is 3 .mu.g/kg/day (effective dose shown in FIG.
10) 3. Therefore, the ratio of itch to EAE in mouse=1 4. Using the
ratio of efficacy for itch vs EAE in the same species (mouse) of 1
5. The effective dose in humans for itch of 2.5 .mu.g/body/day 6.
Calculation to convert EAE mouse to Human dose prediction: EAE
mouse dose/(3 .mu.g/kg/day mouse itch.times.2.5 .mu.g/body/day
human itch)=Human MS dose. 7. Conversion of EAE mouse dose to
predicted human MS dose: 1. 3 .mu.g/kg/day mouse=2.5 .mu.g/body/day
for human (FIG. 10) 2. 10 .mu.g/kg/day mouse=8.33 .mu.g/body/day
for human (FIG. 1) 3. 30 .mu.g/kg/day mouse=25 .mu.g/body/day for
human (FIG. 1) 4. 100 .mu.g/kg/day mouse=83.33 .mu.g/body/day for
human (FIG. 1) 5. 300 .mu.g/kg/day mouse=250 .mu.g/body/day for
human (FIG. 1)
As many demyelinating diseases cause horribly debilitating
symptoms, any improvement in treatment outcomes provides an
important development. The inventors have discovered that
nalfurafine is an effective treatment for demyelinating diseases,
and in particular MS. In one example, the inventors believe that
treatment with nalfurafine will be effective for alleviating the
debilitating symptoms related to Clinically Isolated Syndrome
(CIS). One of the MS disease courses, CIS generally refers to a
first episode of neurologic symptoms associated with MS. Typically,
this initial episode is caused by inflammation or demyelination in
the central nervous system (CNS), and will last 24 hours or
more.
Therefore, in one aspect, the invention provides a method of
treating a demyelinating disease in a subject in need thereof,
comprising administering a therapeutically effective amount of
nalfurafine to the subject.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject comprising identifying a subject
who would benefit from a decreased level of demyelination and
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
The term "treating" as used herein with reference to a disease or
condition refers to the following: (a) ameliorating the disease or
condition such as by eliminating or causing regression of or
decreasing the severity of the disease or medical condition of the
subject being treated relative to an untreated subject according to
art-accepted criteria for monitoring the disease or condition
(Wattjes et al. (2015). Evidence-based guidelines: MAGNIMS
consensus guidelines on the use of MRI in multiple
sclerosis--establishing disease prognosis and monitoring patients.
Nat. Rev. Neurol. 11, 597-606; Traboulsee et al. (2016). Revised
Recommendations of the Consortium of MS Centers Task Force for a
Standardized MRI Protocol and Clinical Guidelines for the Diagnosis
and Follow-Up of Multiple Sclerosis. AJNR Am. 3. Neuroradiol. 37,
394-401; Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13,
83-99; Ontaneda et al. (2017). Clinical outcome measures for
progressive MS trials. Mult. Scler. 23, 1627-1635; Naismith et al.
(2012). Diffusion tensor imaging in acute optic neuropathies:
predictor of clinical outcomes. Arch. Neurol. 69, 65-71); (b)
suppressing the disease or condition such as by slowing or
arresting the development of the disease or condition relative to
an untreated subject according to art-accepted criteria for
monitoring the disease or condition (Oh et al. (2019). Imaging
outcome measures of neuroprotection and repair in MS: A consensus
statement from NAIMS. Neurology; Sormani et al. (2017). Assessing
Repair in Multiple Sclerosis: Outcomes for Phase II Clinical
Trials. Neurother. 3. Am. Soc. Exp. Neurother. 14, 924-933; Zhang
et al. (2018). Clinical trials in multiple sclerosis: milestones.
Ther. Adv. Neurol. Disord. 11; Bjartmar et al. (2003). Axonal loss
in the pathology of MS: consequences for understanding the
progressive phase of the disease. 3. Neurol. Sci. 206, 165-171;
Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13, 83-99) or
(c) alleviating a symptom of the disease or condition in the
subject relative to an untreated subject according to art-accepted
criteria for monitoring the disease or condition (van Munster et
al. (2017). Outcome Measures in Clinical Trials for Multiple
Sclerosis. CNS Drugs 31, 217-236; Uitdehaag (2018). Disability
Outcome Measures in Phase III Clinical Trials in Multiple
Sclerosis. CNS Drugs 32, 543-558; Toosy et al. (2014). Optic
neuritis. Lancet Neurol. 13, 83-99). In some preferred embodiments
"treating" refers to ameliorating as in (a), suppressing as in (b)
and/or alleviating as in (c) in a statistically significant manner
relative to an appropriate untreated control subject according to
art-accepted criteria for monitoring the disease or condition.
In the definition of "treating" the art accepted criteria are one
or more of Criteria for measuring disability may include the
expanded disability scale, multiple sclerosis functional composite
Z-score and multiple sclerosis Impact Scale and Medical Outcomes
Study Short Form, imaging of the brain, spinal cord or optic nerve,
Multiple Sclerosis Functional Composite, and novel composite
measures of disability, in addition to tests evaluating manual
dexterity, ambulation, vision (including measures of axial
diffusivity, visual acuity, contrast sensitivity, visual evoked
potentials (VEPs), and thickness of the retinal nerve fiber layer
(RNFL) and cognition.
The subject may show an observable or measurable decrease in one or
more of the symptoms associated with or related to the disease or
condition as known to those skilled in the art, as indicating
improvement. In some embodiments, the disease or condition is a
demyelinating disease, preferably MS, and the subject shows an
observable and measurable decrease in one or more of the symptoms
associated with or related to MS, preferably a decrease in
demyelination as known to those skilled in the art, as indicating
improvement. In preferred embodiments the improvement is a
statistically significant improvement relative to an appropriate
untreated control subject according to art-accepted criteria for
monitoring the disease or condition.
The terms "decrease" and "reduced" (and grammatical variations
thereof) as used herein with reference to demyelination mean any
measurable or observable reduction in an amount or level of
demyelination or of any symptom of a demyelinating disease that is
attributable to demyelination in a treated subject relative to the
level of demyelination in an appropriate control (e.g., untreated)
subject. In preferred embodiments the measurable or detectable
decrease or reduction is a statistically significant decrease or
reduction, relative to an appropriate control.
The term "increase" (and grammatical variations thereof as used
herein with reference to demyelination means any measurable or
observable increase in an amount or level of remyelination or an
improvement of any symptom of a demyelinating disease that is
attributable to remyelination in a treated subject relative to the
level of remyelination in an appropriate control (e.g., untreated)
subject; e.g., placebo or non-active agent. An example of
quantifying remyelination is demonstrated with treatment with
clemastine fumarate using measures of VEPs to evaluate
remyelination and recovery. (Green et al. (2017) Clemastine
fumarate as a remyelinating therapy for multiple sclerosis
(ReBUILD): a randomised, controlled, double-blind, crossover trial.
Lancet. 390, 2481-2489; Jankowska-Lech et al. (2019). Peripapillary
retinal nerve fiber layer thickness measured by optical coherence
tomography in different clinical subtypes of multiple sclerosis.
Mult. Scler. Relat. Disord. 27, 260-268; Naismith et al. (2012).
Diffusion tensor imaging in acute optic neuropathies: predictor of
clinical outcomes. Arch. Neurol. 69, 65-71; Oh et al. (2019).
Imaging outcome measures of neuroprotection and repair in MS: A
consensus statement from NAIMS. Neurology; Sormani et al. (2017).
Assessing Repair in Multiple Sclerosis: Outcomes for Phase II
Clinical Trials. Neurother. 3. Am. Soc. Exp. Neurother. 14,
924-933. In preferred embodiments the measurable or detectable
reduction is a statistically significant reduction, relative to an
appropriate control.
The terms "administration of" or "administering" should be
understood to mean providing nalfurafine or a pharmaceutical
composition comprising, consisting essentially of, or consisting
of, nalfurafine to the subject in need of treatment in a
therapeutically useful form for the mode of administration.
Nalfurafine can be administered via any suitable route. Potential
routes of administration include without limitation oral,
parenteral (including intramuscular, subcutaneous, intradermal,
intravenous, intraarterial, intramedullary and intrathecal),
intraperitoneal, and topical (including dermal/epicutaneous,
transdermal, mucosal, transmucosal, intranasal (e.g., by nasal
spray or drop), intraocular (e.g., by eye drop), pulmonary (e.g.,
by inhalation), buccal, sublingual, rectal and vaginal.
The term "therapeutically" as used herein means "at disease
onset".
In certain embodiments, nalfurafine is administered via oral dosage
forms such as tablets, capsules, syrups, suspensions, and the like.
In another embodiment, nalfurafine is administered via a
transdermal patch.
The term "therapeutically effective amount" refers to a sufficient
quantity of the active agent, in a suitable composition, and in a
suitable dosage form to treat the noted disease conditions or to
obtain a measurable or observable result such as a decrease in
demyelination or an increase in remyelination. The "therapeutically
effective amount" will vary depending on the compound, the severity
of the demyelination disease, and the species, age, weight, etc.,
of the subject to be treated.
In one embodiment, the therapeutically effective amount of
nalfurafine is the amount equivalent to about 0.003-about 0.3 mg/kg
in a mouse which can be converted according to accepted practice
into an animal or human subject dosage. For example, using the
Reagan-Shaw equation, a therapeutically effective amount of
nalfurafine for a dog would be about 0.67-about 2 mg/kg.
In one embodiment, the therapeutically effective amount of
nalfurafine is the amount equivalent to about 0.003-about 0.3 mg/kg
in a mouse, converted according the method of interspecies
comparison described herein. In one embodiment a therapeutically
effective amount of nalfurafine for a human is about 0.01 to about
5 .mu.g nalfurafine daily, preferably about 0.01 to about 2.5 .mu.g
nalfurafine daily.
In one embodiment the subject is human. In one embodiment the
method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
In one embodiment the method comprises administering about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine daily, preferably less than 1 ug
nalfurafine daily.
In one embodiment the method comprises administering about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
In one embodiment the method comprises a long duration therapy.
In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5 days, at least 6 days,
or at least 7 days.
In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5, preferably at least 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
preferably at least 90 days.
In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least a week, at least 2 weeks,
at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 8
weeks.
In some embodiments the long duration therapy comprises
administration for at least 5 days, at least 6 days, at least 7
days, at least 14 days, for at least 21 days, for at least 28 days,
for at least 35 days, for at least 42 days, for at least 45 days,
for at least 60 days, for at least 120 days, for at least 240 days,
or for at least 360 days.
In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 1 week, at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or at least 52
weeks.
In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 1 month, at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or at least
36 months.
In some embodiments the long duration therapy comprises a dosing
gap, preferably wherein the dosing gap is at least 1 day.
In some embodiments dosing gap comprises at least 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, or 14 days.
In some embodiments the dosing gap comprises at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 weeks.
In some embodiments the dosing gap comprises at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or 11 months.
The term "demyelinating disease" refers to a disease of the nervous
system in which the myelin sheath of neurons is damaged.
Demyelinating diseases include demyelinating myelinoclastic
diseases and demyelinating leukodystrophic diseases. Treatment of a
demyelinating disease can comprise treatment with an agent that
decreases demyelination and/or an agent that increases
remyelination.
Demyelinating diseases may affect the central nervous system and
peripheral nervous system. The central nervous system demyelinating
diseases include multiple sclerosis including clinically isolated
syndrome (CIS) optic neuritis, Devic's disease, inflammatory
demyelinating diseases, central nervous system neuropathies like
those produced by Vitamin B12 deficiency, myelopathies like Tabes
dorsalis, leukoencephalopathies like progressive multifocal
leukoencephalopathy, leukodystrophies, or a combination thereof.
The peripheral nervous system demyelinating diseases include
Guillain-Barre syndrome and its chronic counterpart, chronic
inflammatory demyelinating polyneuropathy, anti-MAG peripheral
neuropathy, Charcot Marie Tooth (CMT) disease, copper deficiency,
progressive inflammatory neuropathy, or a combination thereof. The
term "subject" refers to a mammal, more preferably a human, or
companion animal. Preferred companion animals include cats, dogs
and horses. Other mammalian subjects include agricultural animals,
including horses, pigs, sheep, goats, cows, deer, or fowl: and
laboratory animal, including monkeys, rats, mice, rabbits and
guinea pig.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for treating a demyelinating disease in a subject
in need thereof.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for increasing remyelination in a subject in need
thereof.
The invention also provides nalfurafine for use for treating a
demyelinating disease.
The invention also provides nalfurafine for use for increasing
remyelination.
In one embodiment the disease is a demyelinating myelinoclastic
disease.
In one embodiment the disease is a demyelinating leukodystrophic
disease.
In one embodiment the demyelinating disease is a central nervous
system demyelinating disease. In one embodiment the central nervous
system demyelinating disease is selected from the group comprising
MS (including clinically isolated syndrome; CIS), optic neuritis,
Devic's disease, inflammatory demyelinating diseases, central
nervous system neuropathies, myelopathies like Tabes dorsalis,
leukoencephalopathies, leukodystrophies, or a combination
thereof.
In one embodiment the demyelinating disease is MS.
In another embodiment the demyelinating disease is a peripheral
nervous system demyelinating disease. In one embodiment the
peripheral nervous system demyelinating disease is elected from the
group comprising Guillain-Barre syndrome and its chronic
counterpart, chronic inflammatory demyelinating polyneuropathy,
anti-myelin associated glycoprotein (MAG) peripheral neuropathy,
Charcot Marie Tooth (CMT) disease, copper deficiency and
progressive inflammatory neuropathy.
In another aspect the invention provides a method of increasing
remyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
In another aspect the invention provides a method of increasing
remyelination in a subject comprising identifying a subject who
would benefit from an increased level of remyelination and
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
In another aspect the invention provides a method of increasing
remyelination in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
Specifically contemplated as embodiments of the invention described
herein relating to a method of increasing remyelination in a
subject are all of the embodiments of the invention set forth
herein relating to the aspects of the invention that are methods of
decreasing demyelination, methods of treating MS, methods of
attenuating demyelination, methods of accelerating remission of MS,
and methods of treating a demyelinating disease.
In another aspect the invention provides a method of attenuating
demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject and thereby attenuating a level of demyelination in the
subject relative to the level of demyelination when nalfurafine is
not administered.
In another aspect the invention provides a method of attenuating
demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of an agent that
decreases the level of demyelination in the subject relative to the
level of demyelination before administering the agent and/or that
increases the level of remyelination in the subject relative to the
level of remyelination before administering the agent wherein the
agent comprises nalfurafine.
In one embodiment the subject is human. In one embodiment the
method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
In one embodiment the method comprises administering about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine daily, preferably less than 1 ug
nalfurafine daily.
In one embodiment the method comprises administering about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
The term "attenuation of demyelination" means in certain
embodiments that the amount or level of demyelination in the
subject as a result of the disease or as a symptom of the disease
is reduced when compared to otherwise identical conditions in an
appropriate control subject or at an appropriate control reference
timepoint and/or in certain embodiments that the amount or level of
remyelination in the subject is increased when compared to an
otherwise identical conditions in an appropriate control subject or
at an appropriate control reference timepoint. In some preferred
embodiments the reduction or increase as compared to the
appropriate control is a statistically significant reduction or
increase.
In certain preferred embodiments, the term "attenuation of
demyelination" thus means that the amount of or level demyelination
in the subject as a result of the disease or as a symptom of the
disease is reduced or decreased in a statistically significant
manner when compared to a suitable control as would be understood
by a person of skill in the art in view of the present disclosure
and/or the amount or level of remyelination in the subject is
increased in a statistically significant manner when compared to a
suitable control as would be understood by a person of skill in the
art in view of the present disclosure.
Similarly, the term "improvement in nerve function" refers to a
quantifiable improvement in function having a statistically
different change in a measurable parameter relative to an
appropriate control as recognized by a person of skill in the art.
In some embodiments the improvement in function has a statistically
significant change in the measurable parameter. In one embodiment
the measurable parameter is the disease score as described in
Example 1.
Symptoms attributable to demyelination will vary depending on the
disease but may include, for example but not limited to,
neurological deficits, such as chronic pain, cognitive impairment
(including memory, attention, conceptualization and problem-solving
skills) and information processing; paresthesia in one or more
extremities, in the trunk, or on one side of the face; weakness or
clumsiness of a leg or hand; or visual disturbances, e.g. partial
blindness and pain in one eye (retrobulbar optic neuritis), dimness
of vision, or scotomas.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for attenuating demyelination in a subject in need
thereof.
The invention also provides nalfurafine for use for attenuating
demyelination in a subject in need thereof.
In another aspect the invention provides a method of treating MS in
a subject in need thereof, comprising administering a
therapeutically effective amount of nalfurafine to the subject. The
subject can suffer from any type of MS including CIS, RRMS, PRMS,
SPMS, PRMS or MS that follows a different and/or undefined disease
course.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for treating MS in a subject in need thereof.
The invention also provides nalfurafine for use for treating MS in
a subject in need thereof.
In one embodiment the subject has RRMS. In one embodiment the
subject has PPMS. In one embodiment the subject has, or is
diagnosed as having, SPMS. In one embodiment the subject has, or is
diagnosed as having, PRMS. In one embodiment the subject has, or is
diagnosed as having, Clinically Isolated Syndrome (CIS).
In another aspect the invention provides a method of treating MS in
a subject in need thereof, comprising administering to the subject
a therapeutically effective amount of an agent that decreases a
level of demyelination in the subject relative to the level before
administering the agent and/or that increases a level of
remyelination in the subject in the subject relative to the level
before administering the agent, wherein the agent comprises
nalfurafine.
In some embodiments the methods of treating MS set forth herein can
comprise one or more of the following steps selected from the group
consisting of diagnosing MS in the subject, testing for
demyelination in the subject, testing for a reduction or reversal
in demyelination in the subject, testing for remyelination in the
subject, testing for a level of paralysis or a reduction or
reversal of a level of paralysis in the subject, and testing for a
decrease or increase of coordination and/or balance in the
subject.
In one embodiment a method of treating MS and/or of treating a
demyelinating disease and/or of attenuating demyelination and/or of
increasing remyelination comprises identifying a subject who would
benefit from a level of decreased demyelination.
In some embodiments a subject who would benefit from a level of
decreased demyelination and/or a level of increased remyelination
is identified on the basis of exhibiting one or more clinical
symptoms of MS including, but not limited to: loss of sensitivity
or changes in sensation such as tingling, pins and needles or
numbness, muscle weakness of variable severity, very pronounced
reflexes, muscle spasms, or difficulty in moving; difficulties with
coordination and balance (ataxia); spasticity; problems with speech
or swallowing, visual problems (nystagmus, optic neuritis or double
vision), fatigue, acute or chronic pain, facial pain (trigeminal
neuralgia), bladder and bowel difficulties, incontinence, reduced
cognitive ability, depression, anxiety and other emotional
abnormalities, sexual dysfunction, Uhthoff's phenomenon (a
worsening of symptoms due to exposure to higher than usual
temperatures), and Lhermitte's sign (an electrical sensation that
runs down the back when bending the neck).
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 5 mg nalfurafine daily, about 0.01 to about 4 .mu.g, about
0.01 to about 3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to
about 2 .mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1
.mu.g, about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5
.mu.g, or about 0.25 .mu.g nalfurafine daily.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine daily, preferably less than 1 ug
nalfurafine daily.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
In one embodiment the treatment results in one or more clinical
outcomes as compared to subjects not treated with nalfurafine,
selected from the group consisting of: (a) a decrease in MS disease
progression; (b) a decrease in MS disease severity; (c) a decrease
in nerve cell demyelination; (d) a decrease in frequency or
severity of relapsing MS attacks; (e) a decrease in MS clinical
symptoms; (f) the healing of damaged nerve tissue
(neuro-restoration); (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); (h) the protection of damaged nerve
tissue from further disease activity (neuroprotection); (i) the
promotion neuronal outgrowth (neuro-regeneration) in the central
nervous system; (j) a decrease in disability caused by MS; (k) an
improvement of nerve function; and (l) an enhanced rate of
remission.
In another embodiment the treatment results in a reduction of one
or more clinical symptoms of MS including, but not limited to loss
of sensitivity or changes in sensation such as tingling, pins and
needles or numbness, muscle weakness of variable severity, very
pronounced reflexes, muscle spasms, or difficulty in moving;
difficulties with coordination and balance (ataxia); spasticity;
problems with speech or swallowing, visual problems (nystagmus,
optic neuritis or double vision), fatigue, acute or chronic pain,
facial pain (trigeminal neuralgia), bladder and bowel difficulties,
incontinence, reduced cognitive ability, depression, anxiety and
other emotional abnormalities, sexual dysfunction, Uhthoff's
phenomenon (a worsening of symptoms due to exposure to higher than
usual temperatures), and Lhermitte's sign (an electrical sensation
that runs down the back when bending the neck).
In one aspect the invention provides a method of accelerating
remission of MS in a subject in need thereof, the method comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
In one aspect the invention provides a method of accelerating
remission from MS in a subject in need thereof, the method
comprising administering a therapeutically effective amount of an
agent that decreases the level of demyelination in the subject
relative to the level of demyelination before administering the
agent, wherein the agent comprises nalfurafine.
In one aspect the invention provides a method of accelerating
remission from MS in a subject in need thereof, the method
comprising administering a therapeutically effective amount of an
agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
The invention also provides a use of nalfurafine in the manufacture
of a medicament for accelerating remission from MS in a subject in
need thereof.
The invention also provides nalfurafine for use in accelerating
remission from MS in a subject in need thereof.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 5 .mu.g nalfurafine daily, about 0.01 to about 4 .mu.g, about
0.01 to about 3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to
about 2 .mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1
.mu.g, about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5
.mu.g, or about 0.25 .mu.g nalfurafine daily.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine daily, preferably less than 1 ug
nalfurafine daily.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
The term "enhanced remission of MS" as used herein, means that the
start of the remission process is reached faster and/or the rate at
which remission is achieved is faster (as compared to subjects not
treated with nalfurafine).
Remission of MS can be measured using any technique known in the
art including but not limited to physical disability status,
biological markers and brain scans using MRI.
In one aspect the invention provides a method of treating MS in a
human subject in need thereof, the method comprising administering
to the subject about 0.01 to about 5 mg nalfurafine daily, about
0.05 to about 2.0 mg, about 0.15 to 0.6 mg nalfurafine daily,
wherein the treatment results in one or more clinical outcomes as
compared to subjects not treated with nalfurafine selected from the
group consisting of: (a) a decrease in MS disease progression; (b)
a decrease in MS disease severity; (c) a decrease in nerve cell
demyelination; (d) a decrease in frequency or severity of relapsing
MS attacks; (e) a decrease in MS clinical symptoms; (f) the healing
of damaged nerve tissue (neuro-restoration); (g) an increase in
remyelination of demyelinated nerves in the central nervous system
(neuro-restoration/protection); (h) the protection of damaged nerve
tissue from further disease activity (neuroprotection); (i) the
promotion neuronal outgrowth (neuro-regeneration) in the central
nervous system; (j) a decrease in disability caused by MS; (k) an
improvement of nerve function; and (l) an enhanced rate of
remission.
In one aspect the invention provides a method of treating MS in a
human subject in need thereof, the method comprising administering
to the subject about 0.01 to about 5 .mu.g nalfurafine daily, about
0.01 to about 4 .mu.g, about 0.01 to about 3 .mu.g, about 0.01 to
about 2.5 .mu.g, about 0.01 to about 2 .mu.g, about 0.01 to about
1.5 .mu.g, about 0.01 to about 1 .mu.g, about 0.01 to about 0.75
.mu.g, about 0.01 to about 0.5 .mu.g, or about 0.25 .mu.g
nalfurafine daily, wherein the treatment results in one or more
clinical outcomes as compared to subjects not treated with
nalfurafine selected from the group consisting of: (a) a decrease
in MS disease progression; (b) a decrease in MS disease severity;
(c) a decrease in nerve cell demyelination; (d) a decrease in
frequency or severity of relapsing MS attacks; (e) a decrease in MS
clinical symptoms; (f) the healing of damaged nerve tissue
(neuro-restoration); (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); (h) the protection of damaged nerve
tissue from further disease activity (neuroprotection); (i) the
promotion neuronal outgrowth (neuro-regeneration) in the central
nervous system; (j) a decrease in disability caused by MS; (k) an
improvement of nerve function; and (l) an enhanced rate of
remission.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
In some embodiments the method comprises administering less than
about 1 .mu.g nalfurafine daily, preferably less than 1 ug
nalfurafine daily.
In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
In another aspect the invention provides a method of treating a
demyelinating disease in a subject comprising identifying a subject
who would benefit from a decreased level of demyelination and
administering to the subject a therapeutically effective amount of
an agent that decreases the level of demyelination relative to the
level of demyelination before administering the agent, wherein the
agent comprises nalfurafine.
In another aspect the invention provides a method of increasing
remyelination in a subject comprising identifying a subject who
would benefit from an increased level of remyelination and
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination relative to the
level of remyelination before administering the agent, wherein the
agent comprises nalfurafine.
Specifically contemplated as embodiments of the invention described
herein relating to nalfurafine for use in decreasing demyelination,
attenuating demyelination, accelerating remission of MS, treating
MS, treating a demyelinating disease and increasing remyelination
are all of the embodiments of the invention set forth herein
relating to the aspects of the invention that are methods of
decreasing demyelination, attenuating demyelination, accelerating
remission of MS, treating MS, treating a demyelinating disease and
increasing remyelination.
Additionally, specifically contemplated as embodiments of the
invention described herein relating to the use of nalfurafine in
the manufacture of a medicament for decreasing demyelination,
attenuating demyelination, accelerating remission of MS, treating
MS or for increasing remyelination are all of the embodiments of
the invention set forth herein relating to the aspects of the
invention that are methods of decreasing demyelination, attenuating
demyelination, accelerating remission of MS, treating MS, treating
a demyelinating disease and increasing remyelination.
In addition, specifically contemplated herein for all recited
method, use and nalfurafine for use aspects of the invention are
all of the embodiments set out herein that relate to long duration
therapy and dosing gaps in long duration therapy.
The invention consists in the foregoing and also envisages
constructions of which the following gives examples only and in no
way limit the scope thereof.
6.4 Examples
Example 1: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Therapeutically in the Experimental Autoimmune
Encephalomyelitis (EAE) Model of MS
Experimental detail: Female, C57BL/6 mice were immunized
subcutaneously (s.c.) in the hind flanks to induce EAE using myelin
oligodendrocyte glycoprotein (MOG) peptide 35-55 (50 mg/mouse) in
complete Freund's adjuvant containing heat-killed Mycobacterium
tuberculosis (500 .mu.g/mouse). In addition, pertussis toxin (200
ng/mouse) was administered intraperitoneally (i.p.) on days 0 and
2. Mice were weighed and scored daily. On day 17 (vertical dotted
line in FIG. 1), mice were started on daily treatment with vehicle
only (Veh; 10% tween and 10% DMSO in saline) or nalfurafine at 0.3,
0.1, 0.03, or 0.01 mg/kg by i.p. injection. Nalfurafine was
obtained from the University of Kansas, Synthetic Chemical Biology
Core Laboratory (97.6% pure by HPLC). Treatment allocation was
blinded. The disease was scored from 0-5 with 0 (normal), 1
(partial tail paralysis), 2 (full tail paralysis), 3 (one hind limb
paralysed or severe disability in both hind limbs), 4 (complete
paralysis of both hind limbs) and 5 (moribund). This model is a
standard disease model for multiple sclerosis and is described in
White et al. 2018. Scientific Reports. 8:259 which is incorporated
herein by reference in its entirety. Shown in FIG. 1 are results
combined from 2 independent experiments. **** p<0.0001 &*
p<0.05 by one-way ANOVA with Dunnett's multiple comparison
test.
Interpretation and impact: The results demonstrate that nalfurafine
is able to treat on-going disease. The reduction of disease in all
nalfurafine-treated groups indicates recovery from paralysis, which
is complete at some doses (0.1 and 0.03 mg/kg) and unusual in this
model. Finally, the dose at which nalfurafine shows the most rapid
recovery in this example is 0.1 mg/kg with doses above and below
this level appearing less effective.
Example 2: Nalfurafine Reduces Total Disability when Administered
Therapeutically in the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. On day 17, mice were started on daily
treatment with vehicle only (Veh) or nalfurafine at 0.3, 0.1, or
0.03 mg/kg by i.p. injection. The area under the curve (AUC) was
calculated for each mouse based upon the daily disease score and
represents the total disability experienced. Shown in FIGS. 2 A-B
are results from 1 representative experiment. * p<0.05 by
one-way ANOVA with Dunnett's multiple comparison test.
Interpretation and impact: Despite all treatment groups having
similar disease scores at the start of treatment (lower graph),
mice treated daily with nalfurafine had significantly lower total
disability by day 45 after immunization to induce EAE (upper
graph). Doses of 0.03 and 0.1 mg/kg nalfurafine had the greatest
effect at reducing disability. The 0.1 mg/kg nalfurafine dose
results in a 60% reduction in disease.
Without wishing to be bound by theory, the inventors believe that
the results in Example 2 highlight the benefits of treatment with
nalfurafine over a period of at least a week. Accordingly, in some
embodiment's administration comprises administration for at least 7
days, at least 14 days, at least 30 days, at least 45 days, at
least 60 days, at least 120 days, at least 240 days, or at least
360 days.
Example 3: Nalfurafine Promotes Recovery from EAE-Induced Weight
Loss when Administered Therapeutically
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Mice were weighed daily and the % change in
body weight calculated. On day 17 (vertical dotted line in FIG. 3),
mice were started on daily treatment with vehicle only (Veh) or
nalfurafine at 0.3, 0.1, or 0.03 mg/kg by i.p. injection.
Interpretation and impact: As shown in FIG. 3, at onset of disease,
mice rapidly lose weight. Once treatment with nalfurafine is
initiated (vertical dotted line), mice recover from EAE-induced
weight loss.
Example 4: Nalfurafine Reduces the Immune Cell Infiltration into
the Brain when Administered at Low Doses Therapeutically in the EAE
Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. On day 17, mice were started on daily
treatment with vehicle only (Veh) or nalfurafine at 0.3, 0.1, or
0.03 mg/kg by i.p. injection. On day 45 after immunization to
induce EAE, mice were culled, and immune cells isolated from the
brains. Isolation was by Percoll gradient as described in White et
al. 2018. Scientific Reports. 8:259. Once isolated, cells were
stained with fluorescently labelled antibodies to identify specific
immune cell types and analysed by flow cytometry. All infiltrating
immune cells were identified by CD45.sup.high expression; CD4 T
cells were identified as CD45.sup.highCD4+, and macrophages as
CD45.sup.highCD11b.sup.+Gr-1.sup.-. The relative number of cells is
expressed as a ratio to microglia (MG), a brain resident immune
cell identified as CD45.sup.mediumCD11b.sup.+. * p<0.05 by
one-way ANOVA with Dunnett's multiple comparison test.
Interpretation and impact: As shown in FIGS. 4A-C, at day 45, there
was a significant elevation in immune cells in the brains of
vehicle-treated EAE mice compared to healthy animals. Treatment
with 0.03 mg/kg nalfurafine significantly reduced the number of
infiltrating immune cells suggesting that at this dose, nalfurafine
can have immunomodulatory properties. Interestingly, while mice
treated with 0.1 nalfurafine had similar levels of infiltrating
cells as vehicle-treated animals, these mice had no overt signs of
disease and had recovered fully from paralysis (FIG. 1).
Example 5: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Before the Onset of Paralysis in the EAE Model of
MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. On day 17 (vertical dotted line in FIG. 5),
mice were started on daily treatment with vehicle only (Veh) or
nalfurafine at 0.3, 0.1, or 0.03 mg/kg by i.p. injection. Shown in
FIG. 5 are results in mice that were not sick at the time of
treatment but developed disease later. * p<0.05 by two-way ANOVA
with Holm-Sidak's multiple comparison test.
Interpretation and impact: Treating with nalfurafine prior to
disease onset did not alter the onset of disease. However,
treatment with nalfurafine led to a rapid recovery from paralysis
compared to vehicle-treated mice. These data suggest that treating
with nalfurafine will also be effective at reducing total
disability if administered before disease but may not prevent
onset.
Example 6: Myelination is Improved in Mice Treated with Nalfurafine
after the Onset of Paralysis in the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. On day 17, mice were started on daily
treatment with vehicle only or nalfurafine at 0.03 mg/kg by i.p.
injection. On day 45 after immunization to induce EAE, mice were
culled, and spinal cords were processed for transmission electron
microscopy (TEM). Shown in FIGS. 6A-C are representative TEM images
of spinal cord sections from a healthy (A), vehicle-treated EAE
(B), or nalfurafine-treated EAE mouse (C) stained to show that dark
myelin rings around the nerve axons.
Interpretation and impact: At day 45, there was a significant
reduction in the dark stained myelin in the spinal cord of the
vehicle-treated EAE mice suggesting demyelination has occurred.
Additionally, the nerve axons appear bloated and the cytoplasm
disorganized suggesting cellular stress. In contrast, the nerve
axons appear healthy and well-myelinated in the nalfurafine-treated
mouse, which is concordant with full functional recovery.
Example 7: Nalfurafine Improved Weight Gain when Administered after
Demyelination in the Cuprizone Model of Demyelination
Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone
in the diet for 5 weeks to induce demyelination. At the start of
week 4 (vertical dashed line in FIG. 7), mice were started on daily
treatment with vehicle only or nalfurafine 0.1 mg/kg by i.p.
injection. At the start of week 5 (vertical dotted line in FIG. 7),
cuprizone was removed from the diet to enable spontaneous
remyelination. Mice were weighed daily and the % weight change
calculated.
Interpretation and impact: As shown in FIG. 7, cuprizone caused
significant weight loss in mice as previously reported. This weight
loss was reversed significantly more effectively by administration
of nalfurafine than vehicle alone.
Example 8: Nalfurafine Enhances the Functional Recovery of
Coordination and Balance when Administered after Demyelination in
the Cuprizone Model of Demyelination
Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone
in the diet for 5 weeks to induce demyelination and treated with
nalfurafine as described in Example 7. Behavioural tests including
the rotarod assay, which measures coordination, were performed
weekly. Mice were trained on an accelerating rotarod apparatus
(Panlab, Harvard Apparatus) over a period of 4 to 5 days before
recording baseline latencies at day 0 followed by weekly
measurements throughout cuprizone treatment and recovery. The
rotarod was set to 4 rotations per minute (rpm) and an acceleration
rate of 40 rpm with a maximum cut-off time of 5 minutes. The time
and speed at which the animal falls off the rotating rod was
recorded and the average of 3 replicates recorded. Data shows
performance at week 9 following Veh or nalfurafine (0.1 mg/kg)
treatment relative to performance at week 5. * p<0.05 by
Students t-test.
Interpretation and impact: As shown in FIG. 8, cuprizone impaired
coordination in mice as previously reported. Cuprizone-induced
disability was reversed by administration of nalfurafine. These
data suggest that nalfurafine is effective at reducing disability
in a model of non-immune mediated demyelination such as that found
in some progressive MS patients.
Example 9: Nalfurafine Enhances Myelination when Administered after
Demyelination in the Cuprizone Model of Demyelination
Experimental detail: Female, C57BL/6 mice were fed 0.3% cuprizone
in the diet for 5 weeks to induce demyelination as described in
Example 7. On day 65, mice were culled, and brains were processed
for transmission electron microscopy (TEM). Shown are
representative TEM images of sections from the corpus callosum of a
healthy (no cuprizone), vehicle-treated & cuprizone-treated, or
nalfurafine-treated & cuprizone-treated mouse stained to show
the dark myelin rings around the nerve axons. Myelin was quantified
by g-ratio, which is the inner axonal diameter divided by the total
outer diameter.
Interpretation and impact: As shown in FIGS. 9 A-D, cuprizone
caused a loss of myelin and a concurrent disruption in regular
axonal structures in the corpus callosum compared to healthy
controls. In contrast, more myelin was detected, and the structure
was less disorganized in the corpus callosum of animals treated
with cuprizone & nalfurafine. These data indicate that
nalfurafine treatment promotes remyelination and repair after
cuprizone-induced, non-immune-mediated demyelination. A similar
non-immune associated demyelination occurs in some progressive MS
patients.
Example 10: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Therapeutically in the Experimental Autoimmune
Encephalomyelitis (EAE) Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 10. On the day of
disease onset (score .gtoreq.1, dotted line), mice were started on
daily treatment with vehicle only (Veh) or nalfurafine at 0.3, 0.1,
0.03, 0.01, or 0.003 mg/kg by i.p. injection. Treatment allocation
was blinded. Shown are the aligned scores from mice (n=33 in Veh, 3
in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg
groups) starting from onset/treatment initiation. One animal in the
0.3 mg/kg nalfurafine group and 2 from the vehicle group were
euthanized at day 17-18. ****p<0.0001 by two-way ANOVA all doses
(except 0.3 mg/kg) compared to vehicle.
Interpretation and impact: By treating after the onset of disease
(paralysis), we show that nalfurafine is able to treat on-going
disease. The reduction of disease in all nalfurafine-treated groups
indicates recovery from paralysis, which is complete at some doses
(0.01 and 0.03 mg/kg); full recovery from disease is unusual in
this model and the efficacy of the nalfurafine treatment is
surprising. Finally, the dose at which nalfurafine shows the most
rapid recovery in this example is 0.01 mg/kg, and this finding has
been replicated in 6 independent experiments.
Example 11: Nalfurafine is not Effective when Administered as a
Short 4-Day Course Starting at Disease Onset in EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 11. On the day of
disease onset (score .gtoreq.1, dotted line), mice were started on
daily treatment with vehicle only or nalfurafine at 0.01 mg/kg by
i.p. injection daily throughout the experimental course or only for
four days (shaded area). Shown are the aligned scores from mice
(n=5/group) starting from onset/treatment initiation. **p<0.01
by two-way ANOVA NalF (full treatment) compared to nalfurafine (4
days) or vehicle.
Interpretation and impact: Treatment with nalfurafine does not
enhance recovery when administered for only four days starting from
disease onset, whereas treatment with a longer duration does
enhance recovery effectively.
Example 12: Nalfurafine does not Alter Peak Disease when
Administered Therapeutically in the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 12. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01,
or 0.003 mg/kg by i.p. injection. The peak disease score during the
first EAE episode was recorded and shown are the mean and standard
error of individual mice (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in
0.03, 20 in 0.01, and 4 in 0.003 mg/kg groups). No significant
differences were found between any nalfurafine dose and vehicle by
Kruskal-Wallis with Dunn's multiple comparison test. These data are
from the same experiments as Example 10.
Interpretation and impact: Because no difference in peak disease
score was found at any dose of nalfurafine compared to vehicle,
nalfurafine did not appear to alter the initial immune-mediated
neuroinflammatory event that leads to demyelination and paralysis.
This finding suggests that the functional improvement observed
(i.e. the recovery from paralysis) occurs because the initial
insult has been repaired and perhaps not because the initial insult
itself was stopped.
Example 13: Nalfurafine Promotes Full Recovery from EAE-Induced
Paralysis when Administered Therapeutically
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 13. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01,
or 0.003 mg/kg by i.p. injection. Mice were considered recovered if
they received a score .ltoreq.0.5 by day 23 post treatment
initiation. Shown are the percentages of mice in each group that
recovered (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01,
and 4 in 0.003 mg/kg groups). ****p<0.0001, **p<0.01, and
*p<0.05 by Fisher's exact test. These data are from the same
experiments as Example 10.
Interpretation and impact: Treatment with nalfurafine enables full
functional recovery (i.e. no paralysis) when administered
therapeutically and at a wide range of doses (0.003-0.1 mg/kg all
show a significant effect). Full recovery in this model of disease
is unusual. The efficacy achieved with the treatment of nalfurafine
is extraordinary.
Example 14: Nalfurafine Promotes Full Recovery from EAE-Induced
Paralysis when Administered Therapeutically with an EC.sub.50 of
<0.001 Ma/Kg
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 14. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.1, 0.03, 0.01, or
0.003 mg/kg by i.p. injection. Mice were considered recovered if
they received a score .ltoreq.0.5 by day 23 post treatment
initiation. Shown are the percentages of mice in each group that
recovered (n=33 in Veh, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in
0.003 mg/kg groups). A dose-response curve has been fitted from a
dose of 0.1 mg/kg, in which 100% recovered, to the vehicle alone,
in which 12.1% recovered. This curve calculates an EC.sub.50 of
<0.001 mg/kg. These data are from the same experiments as
Example 13.
Interpretation and impact: Treatment with Nalfurafine enables full
functional recovery (i.e. no paralysis) when administered
therapeutically and at a wide range of doses (0.003-0.1 mg/kg all
show a significant effect). Full recovery in this model of disease
is unusual. The efficacy achieved with the treatment of nalfurafine
is extraordinary. To achieve 50% of this effect (i.e. EC.sub.50) an
estimated dose of <0.001 mg/kg is required.
Example 15: Nalfurafine Promotes Sustained Functional Recovery from
EAE-Induced Paralysis when Administered Therapeutically
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1, Results are shown in FIG. 15. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01,
or 0.003 mg/kg by i.p. injection. Mice were considered recovered if
they received a score .ltoreq.0.5 by day 23 post treatment
initiation. Shown are the number of days mice were in recovery in
each group (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01,
and 4 in 0.003 mg/kg groups). ****p<0.0001, **p<0.01, and
*p<0.05 by one-way ANOVA with Holm-Sidak's multiple comparison
test. These data are from the same experiments as Example 10.
Interpretation and impact: Treatment with nalfurafine enables a
sustained functional recovery (i.e. no paralysis) when administered
therapeutically and at a wide range of doses (0.003-0.1 mg/kg all
show a significant effect).
Example 16: Nalfurafine Promotes Functional Recovery from Paralysis
in Male Mice when Administered Therapeutically in EAE Model of
MS
Experimental detail: EAE was induced in male C57BL/6 mice as
described in Example 1. Results are shown in FIG. 16. On the day of
disease onset (score .gtoreq.1, line), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection. Treatment allocation was blinded. Shown are the aligned
scores from mice (n=5/group) starting from onset/treatment
initiation. ****p<0.0001 by two-way ANOVA compared to
vehicle.
Interpretation and impact: Nalfurafine is effective at enabling
functional recovery from paralysis in both females and males.
Example 17: Nalfurafine Promotes Full Recovery in Male Mice when
Administered Therapeutically in EAE Model of MS
Experimental detail: EAE was induced in male C57BL/6 mice as
described in Example 1. Results are shown in FIG. 17. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection. Mice were considered recovered if they received a score
.ltoreq.0.5 by day 23 post treatment initiation. Shown are the
percentages of mice in each group that recovered (n=5/group).
**p<0.01 by Fisher's exact test. These data are from the same
experiments as Example 16.
Interpretation and impact: Treatment with nalfurafine promotes full
recovery (i.e. no paralysis) in both female and male when
administered therapeutically.
Example 18: Nalfurafine Promotes Sustained Recovery in Male Mice
from EAE-Induced Paralysis when Administered Therapeutically
Experimental detail: EAE was induced in male C57BL/6 mice as
described in Example 1. Results are shown in FIG. 18. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection. Mice were considered recovered if they received a score
.ltoreq.0.5 by day 23 post treatment initiation. Shown are the
number of days mice were in recovery in each group (n=5/group).
****p<0.0001 by Student's t test. These data are from the same
experiments as Example 16.
Interpretation and impact: Treatment with nalfurafine enables a
sustained functional recovery (i.e. no paralysis) in both females
and males when administered therapeutically.
Example 19: Nalfurafine Treatment Reduces the Immune Cell
Infiltration into the Brain when Administered Therapeutically in
the EAE Model of MS (A) Whereas U-50488 does not (B)
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 19A and 19B. On
the day of disease onset (score .gtoreq.1), mice were started on
daily treatment with vehicle only or nalfurafine at 0.3, 0.1, 0.03,
0.01, or 0.003 mg/kg by i.p. injection (A). In a separate
experiment, mice were similarly treated with vehicle alone or
U-50488, a KOR agonist at 1.6 and 5 mg/kg (B). During the chronic
phase (>24 days post treatment initiation), mice were culled,
and immune cells isolated from the brains. Isolation was by Percoll
gradient as described in White et al. 2018. Scientific Reports.
8:259. Once isolated, cells were stained with fluorescently
labelled antibodies to identify specific immune cell types and
analysed by flow cytometry. All infiltrating immune cells were
identified by CD45.sup.high expression. The relative number of
cells is expressed as a ratio to microglia (MG), a brain resident
immune cell identified as CD45.sup.mediumCD11b.sup.+. *p<0.05 by
one-way ANOVA with Holm-Sidak's multiple comparison test compared
to vehicle. NS, not-significant.
Interpretation and impact: In the chronic stage of EAE, there was a
significant elevation in immune cells in the brains of
vehicle-treated EAE mice compared to healthy animals (A). Treatment
with 0.03 and 0.01 mg/kg nalfurafine significantly reduced the
number of infiltrating immune cells suggesting that at these doses,
nalfurafine can have immunomodulatory properties. Interestingly,
while mice treated with 0.1 and 0.003 nalfurafine had similar
levels of infiltrating cells as vehicle-treated animals, these mice
had no overt signs of disease and had recovered fully from
paralysis (FIG. 13). Additionally, nalfurafine but not U-50488
reduced neuroinflammation in this model indicating that not all KOR
agonists have this activity (B).
Example 20: Myelination is Improved in Mice Treated with
Nalfurafine after the Onset of Paralysis in the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 20. On the day of
disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.03 or 0.01 mg/kg by
i.p. injection. During the chronic phase (>24 days post
treatment initiation), mice were culled, perfused with 4%
paraformaldehyde and spinal cords were processed for histology.
Sections were stained with luxol fast blue to assess the % area of
the spinal cord that is demyelinated (i.e. does not stain with
luxol fast blue). % demyelination was assessed using ImageJ. Shown
are the means and standard error of individual values from vehicle
(n=7) or 0.01 (n=6) and 0.03 (n=4) nalfurafine-treated EAE mice.
**p<0.01 by one-way ANOVA with Holm-Sidak's multiple comparison
test.
Interpretation and impact: During the chronic phase, when
nalfurafine enabled full functional recovery in mice, there was a
significant reduction in the percentage of demyelination in the
spinal cord of the nalfurafine-treated EAE mice suggesting
remyelination may have occurred.
Example 21: Nalfurafine does not Alter the Proportion of Major
Lymphocyte Populations in the Spleen During the Chronic Phase of
EAE
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 21A-C. On the
day of disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection. During the chronic phase (27 days post treatment
initiation), mice were culled and their splenocytes assessed by
flow cytometry. The percentage of the major lymphocyte populations
were identified using CD4 (CD4 T helper cells), CD8 (CD8 cytotoxic
T cells), and B220 (B cells), and expressed as % live leukocytes
(i.e. CD45+ cells). Shown are the means and standard error of
individual mice with n=3 (healthy), 4 (vehicle) and 8. No
significant differences were found between vehicle and healthy or
nalfurafine by one-way ANOVA with Holm-Sidak's multiple comparison
test.
Interpretation and impact: Nalfurafine do not alter the proportion
of the major lymphocyte populations in the spleen despite reducing
the number of infiltrating immune cells into the central nervous
system. The maintenance of normal lymphocyte numbers in the spleen
in the nalfurafine treated mice indicates that nalfurafine does not
reduce immune cell infiltration into the brain by killing immune
cells.
Example 22: Nalfurafine does not Alter the Overall Number of CD4 T
Helper Cells in the Spleen but Shifts the CD4 T Cells from an
Effector to Memory State being Suggestive of Immune Resolution
During the Chronic Phase of EAE
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 22A-D. On the
day of disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection. During the chronic phase (27 days post treatment
initiation), mice were culled and their splenocytes assessed by
flow cytometry. Naive CD4 T cells
(CD4.sup.+CD44.sup.-CD62L.sup.high), effector CD4 T cells
(CD4.sup.+CD44.sup.+CD62L.sup.-), and central memory CD4 T cells
(CD4.sup.+CD44.sup.+CD62L.sup.high) are expressed as % CD4 T cells.
"Teff:cm ratio" is the ratio of effector to central memory T cells.
Shown are the means and standard error of individual mice with n=3
(healthy), 4 (vehicle) and 8. **p<0.01 and *p<0.05 by one-way
ANOVA with Holm-Sidak's multiple comparison test.
Interpretation and impact: The increased effector to central memory
ratio in the vehicle-treated mice with EAE compared to healthy mice
indicates an on-going and active immune response mediated by CD4 T
cells. The overall number of CD4 T cells was the same between
nalfurafine and vehicle treated mice. The reduced ratio in the
nalfurafine-treated compared to the vehicle-treated mice indicates
a shift toward a memory phenotype which occurs during the
resolution phase of the immune response. The shift to a memory
state indicates that immune resolution is occurring in
nalfurafine-treated mice in a model of MS where disease is driven
by an active immune response.
Example 23: Nalfurafine Reduces Disease but does not Enable Full
Recovery when the Kappa Opioid Receptor (KOR) is Blocked
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIG. 23. On the day of
disease onset (score >1, dotted line), mice were treated with
vehicle only (daily), nalfurafine (0.01 mg/kg by i.p. injection
daily), the KOR antagonist norBNI (10 mg/kg by i.p. injection
weekly), or both nalfurafine and norBNI. Shown are the aligned
scores from mice (n=8-9/group) starting from onset/treatment
initiation. ****p<0.0001 by two-way ANOVA NalF compared to
vehicle or NalF+noBNI.
Interpretation and impact: Administration of the KOR antagonist,
norBNI, abolishes the ability of nalfurafine to enable full
recovery from paralysis (i.e. score <0.5), and this finding
indicates that the KOR is required for the full effect of
nalfurafine. The finding that nalfurafine is effective at reducing
disease independently of the KOR (i.e. in the presence of norBNI)
indicates that the full mechanism by which nalfurafine exerts its
effects is more complex than KOR activation.
Example 24: Activation of the KOR is Required for Full Recovery
from Paralysis Mediated by Nalfurafine
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 24A-C. On the
day of disease onset (score >1, dotted line), mice were treated
with vehicle only (daily), nalfurafine (0.01 mg/kg by i.p.
injection daily), the KOR antagonist norBNI (10 mg/kg by i.p.
injection weekly), or both nalfurafine and norBNI. The peak disease
score during the first EAE episode was recorded, and mice were
considered recovered if they received a score <0.5 by day 23
post treatment initiation. Shown are the peak disease scores, the
percentage of mice in each group that recovered, and the number of
days in recovery (n=8-9/group). **p<0.01 and ****p<0.0001 by
Fisher's exact test (% recovered) or one-way ANOVA with
Holm-Sidak's multiple comparison test (#days in recovery). These
data are from the same experiments as Example 23.
Interpretation and impact: Administration of the KOR antagonist,
norBNI, abolishes the ability of nalfurafine to enable and sustain
recovery from paralysis (i.e. score <0.5), and this finding
indicates that the KOR is required for the full effect of
nalfurafine at promoting full recovery but not disease
reduction.
Example 25: Myelination is Improved in Mice Treated with
Nalfurafine after the Onset of Paralysis in the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 25A-D. On the
day of disease onset (score .gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine 0.01 mg/kg by i.p.
injection. During the chronic phase (>24 days post treatment
initiation), mice were culled, perfused with 4% paraformaldehyde
and spinal cords were processed for histology. Sections were
stained with luxol fast blue to assess demyelination. The region of
interest taken for analysis is shown in 25A. Note the presence of
demyelinated regions (lesions) with less luxol fast blue (LFB)
staining (myelin) in the ventral horn in EAE mice receiving vehicle
(circle--25B) and no demyelinated lesions present in mice treated
with nalfurafine (25C). Quantified data is shown in 25D. For each
image, 5 randomised regions of the ventral horn of the spinal cord
were analysed in ImageJ using mean grey value and integrated pixel
density as an indicator of myelin density. Data is from two
individual experiments with n=4 (vehicle), n=8 (nalfurafine) EAE
mice respectively. Scale bar=50 .mu.m. *p<0.05 by students
t-test.
Interpretation and impact: EAE disease induces extensive lesions in
the spinal cord (see vehicle only (25B), characterised by a loss of
myelin and neurodegeneration, demonstrating that EAE is a
destructive disease in the CNS. Treatment of this disease state
with nalfurafine reduces this lesion load and demyelination,
suggesting that treatment restores the spinal cord tissue to a near
normal state by remyelination.
Example 26: Nalfurafine Treatment Decreases Cellular Infiltration
into the Spinal Cord when Administered Therapeutically in the EAE
Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 26A-C. On the
day of disease onset (score >1), mice were started on daily
treatment with vehicle only or nalfurafine 0.01 mg/kg by i.p.
injection. During the chronic phase (>24 days post treatment
initiation), mice were culled, perfused with 4% paraformaldehyde
and spinal cords were paraffin embedded for histology. 10 .mu.M
coronal sections were stained with Hematoxylin and Eosin (H&E)
to assess of leucocyte infiltration, a marker of inflammation
within lesions induced in EAE disease. Note the large number of
leucocytes present in the ventral horn of vehicle treated EAE mice,
than in EAE mice administered nalfurafine. Images were scored by a
blinded observer for the level of infiltration on a scale ranging
from 0 (no infiltration) to 3 (maximum infiltration). Data is from
two individual experiments: n=7 mice (11 sections) for EAE Vehicle;
and n=9 mice (13 sections) for EAE mice treated with nalfurafine.
Scale bar=50 .mu.m). Students t-test, *p<0.05.
Interpretation and impact: EAE disease induces substantial
histopathology in the spinal cord. H&E staining of leucocytes
is an indicator of lesion severity, with the higher number of
infiltrating cells, the more severe the lesion, including
demyelination, as shown in the vehicle only panel and by
quantification. Treatment with nalfurafine shows a surprising
reduction of infiltrating leucocytes, with a near absence of
lesions and demyelination indicating that treatment may resolve
lesions and/or cause remyelination.
Example 27: Nalfurafine Treatment Reduces the Level of Activated
Astrocytes in the Spinal Cord when Administered Therapeutically in
the EAE Model of MS
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 27A-1, and 27A-2
day 17, mice were started on daily treatment with vehicle only or
nalfurafine at 0.01 mg/kg by i.p. injection. On day 45 after
immunization to induce EAE, mice were culled, and spinal cords were
processed for immunohistochemistry (IHC). Shown in FIGS. 27A-1 and
27A-2 are representative glial fibiliary acid protein (GFAP)
immunolabeled cells (black staining) from coronal sections of the
ventral horn of the spinal cord taken from EAE mice. The images are
from 10 .mu.M paraffin embedded sections, stained with Rabbit
anti-GFAP at (1:1000, DAKO) before being photographed at 20.times.
magnification, scale bar=50 .mu.m. The number of astrocytes per
section in a standard ROI were counted using the cell counter
plug-in in ImageJ. Two sections were assessed per animal. Sections
assessed n=7 (10-13 sections, from 2 individual experiment.
***p=0.0003 (FIG. 27B).
Interpretation and impact: As shown and quantified in FIGS. 27A-1
and A-2, at day 45, there was significant elevation in the
activated GFAP+ astrocytes in the spinal cord of vehicle treated
EAE mice. Astrocytes are recognized to be early and highly active
players during lesion formation and key for providing peripheral
immune cells access to the central nervous system (Ponath et al.
The Role of Astrocytes in Multiple Sclerosis. Front Immunol. 2018;
9: 217). Treatment with 0.01 mg/kg i.p. nalfurafine significantly
reduces the number of activated astrocytes suggesting that
nalfurafine treatment can have a neuroprotective and
anti-inflammatory effect on the spinal cord tissue in the disease
state (FIG. 27B).
Example 28: Nalfurafine Treatment Enhances Recovery from Weight
Loss when Administered Therapeutically in the Cuprizone
Demyelination Disease Model of MS
FIG. 28A shows a time course of cuprizone induced demyelination and
treatment regime.
Experimental details: A demyelinating disease state was induced in
female C57BL/6 mice (8-14 weeks old and between 17-23 grams in
weight). As shown in the timeline of FIG. 28A, the mice were fed
cuprizone-containing chow (0.3% (w/w) cuprizone) or chow only
(normal controls) for 35 days, at which point they were switched
back to standard chow. At day 28, mice were started on daily
treatment with vehicle only (DMSO: Tween 80: Saline) or nalfurafine
at 0.1 mg/kg by i.p. injection or U-50488 at 1.6 mg/kg by i.p.
injection. On day 70, mice were culled and brain tissue were
processed for transmission electron microscopy (TEM). Mice were
weighed daily and the % weight change calculated.
Interpretation and impact: This model is well established as a tool
for the study of non-immune system induced demyelination. This
model enables the assessment of putative remyelination-promoting
therapeutics (Matsushima and Morell, 2001. The neurotoxicant,
cuprizone, as a model to study demyelination and remyelination in
the central nervous system. Brain Pathol. 11, 107-116).
FIG. 28B shows cuprizone induced weight change over the time course
of study.
Experimental details: A demyelinating disease state was induced in
female C57BL/6 mice as described in Example 28 and illustrated in
FIGS. 28A-C.
Interpretation and impact: Mice treated with 0.3% cuprizone (CPZ)
lose weight as the disease is induced, compared to mice with normal
diet, corresponding to disease induction and severity.
FIG. 28C shows that nalfurafine treatment enhances weight gain in
the recovery phase of the cuprizone demyelination disease model of
MS, whereas U-50488 does not.
Experimental details: A demyelinating disease state was induced in
female C57BL/6 mice as described in FIG. 28C. Diseased animals were
treated with Vehicle only, nalfurafine (0.1 mg/kg), U-50488 (1.6
mg/kg) as described in FIG. 28A. Mice were weighed daily and the %
weight change calculated. *p<0.05=nalfurafine treated mice;
#p<0.05=U-50488 treated mice. Two-way repeated measures ANOVA,
followed by Turkey's multiple comparison tests. (n=15 mice/group
from 3 experimental replicates. ANOVA revealed a significant
interaction F(40, 600)=2.212 (p<0.0001) with significant time
F(8, 600)=101.2 (p<0.0001) and treatment effects F(5,75)=5.52
(P<0.0002).
Interpretation and impact: Mice treated with 0.3% cuprizone (CPZ)
lose weight as the disease is induced. Mice recover when returned
to normal chow (removal of cuprizone) (FIG. 28C). Treatment with
nalfurafine enhances recovery of the lost weight faster compared to
mice with vehicle only or treatment with U-50488.
Example 29: Nalfurafine Treatment Enhances Remyelination when
Administered after Demyelination in the Cuprizone Demyelination
Disease Model of MS
Experimental details: A demyelinating disease state was induced in
female C57BL/6 mice as illustrated in FIG. 28A. The results are
shown in FIGS. 29A-G. Panels A-D of FIG. 29 show representative
Transmission Electron Microscopy (TEM) images of the corpus
callosum of mice (A) fed normal diet and (B-D) fed 0.3% cuprizone
to induce demyelination. Following the time course shown in FIG.
28A, cuprizone fed mice were administered (B) vehicle only
treatment, (C) U-50488 (1.6 mg/kg/i.p.) and (D) nalfurafine (0.1
mg/kg/i.p.) and then sacrificed on experimental day 70. Scale bars
represent 2000 nm.
FIG. 29E shows the quantification and analysis of the g-ratios
shows a significant difference between treatment groups
F(3,953)=21.18 (p<0.0001). Mice fed a normal diet have a mean
g-ratio of 0.78.+-.0.09 in contrast to mice fed 0.3% cuprizone that
have a significant increase in g-ratio of 0.84.+-.0.1 corresponding
to the decreased myelin thickness (####p<0.0001). Mice fed a
diet with cuprizone treated with nalfurafine (0.1 mg/kg/i.p.)
(0.75.+-.0.15) show a significant reduction in g-ratio compared to
Vehicle treated controls (****p<0.0001), corresponding to an
increased myelin thickness. Mice fed a diet with cuprizone treated
with U-50488 show a somewhat increased myelin thickness compared to
vehicle-treated controls with a mean g-ratio of (0.80)
(**p<0.01), but, surprisingly, nalfurafine treatment showed a
significant increase in myelin thickness (decrease in g-ratio)
compared to mice treated with U-50488 (1.6 mg/kg/i.p.) ({circumflex
over ( )}{circumflex over ( )}{circumflex over ( )}p<0.001),
indicating that nalfurafine is significantly more effective at
increasing myelin thickness than U-50488. Data represents
measurements of 5 TEM images of the corpus callosum from two-three
mice per treatment group and g-ratios calculated (a measure of
myelin thickness) using Image J software. Analysis was performed by
individuals blinded to treatment groups. (n=204-267 axons per
treatment group).
FIG. 29F shows the quantification and analysis of the number of
myelinated axons vs non-myelinated axons in a region of interest
(390 .mu.m.sup.2). n=20 images per treatment group (from n=2-3
mice).
FIG. 29G shows the quantification and analysis of the area of
myelin staining per TEM image was performed using Image 3 software
(20 images per treatment from n=2-3 mice sacrificed on day 70). TEM
images were colour inverted (myelin white) and a threshold used to
reveal myelin. The area of this myelin threshold measured for each
treatment group.
All data analysed by one-way ANOVA followed by Turkeys multiple
comparisons test. Significant differences compared to vehicle only
are depicted by *; between normal mice and cuprizone/vehicle
treated mice #; and between nalfurafine and U-50488 by {circumflex
over ( )}. (*p<0.05; **p<0.01; ***p<0.001;
****P<0.0001).
Interpretation and impact: As shown in FIGS. 29A-G, demyelination
was very apparent in the corpus collosum of the brain of
cuprizone-induced, vehicle only treated animals (Panel B). The
ratio between axonal circumference and myelin circumference
(g-ratio) decreases with normal myelination. The cuprizone induced
animals treated with nalfurafine show a more normal axonal-myelin
structure, the myelinated axons are densely packed within white
matter and the myelin sheaths of neighboring fibers often directly
touch. The staining of the myelin sheaths (black) is more prominent
indicating increased remyelination. Ultrastructurally, this
nalfurafine tissue is surprisingly similar to that of the naive
(normal) tissue. Quantitatively, the nalfurafine tissue has a
significantly lower g-ratio compared to vehicle only treated
indicative of enhanced remyelination, with a g-ratio closer to that
of naive (normal) tissue. This is further supported by analysis of
the percentage increase in the number of myelinated axons and
percentage increase in area of myelination in the nalfurafine
treated animals. In contrast, treatment with the compound U-50488
did not show repair or restoration to a near normal state.
Qualitatively, the axonal-myelin structure is disorganised, there
is a loss of axons, and overtly enlarged axonal-myelin structures.
Quantitatively, U-50488 treatment has outcomes similar to that of
the vehicle only treated samples (i.e. no discernible
remyelination), whereas nalfurafine treatment shows similar
outcomes to the naive tissue. Qualitatively and quantitatively,
nalfurafine treatment enhances remyelination that is indicative of
a near-full recovery following a demyelination insult of
cuprizone.
Example 30: Nalfurafine is More Effective at Promoting Functional
Recovery than Clemastine Fumarate, a Known Remyelinating Drug
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 30A-B. On the
day of disease onset (score >1, dotted line), mice were treated
with vehicle only (daily, n=9)) or nalfurafine (0.01 mg/kg by i.p.
injection daily; n=8). In a separate similar experiment, mice were
treated with vehicle only (n=5) or clemastine fumarate (10 mg/kg by
i.p. injection; n=7). Shown are the aligned scores from mice
starting from onset/treatment initiation. ****p<0.0001 by
two-way ANOVA NalF or clemastine compared to vehicle.
Interpretation and impact: Clemastine fumarate, an anti-histamine
which also antagonizes the muscarinic receptor, has been shown to
reduce chronic disability in the EAE model when used at 10 mg/kg
starting at the time of immunization. Additionally, it has been
shown to enhance remyelination in mice and humans (Li et al. 2015,
Clemastine rescues behavioral changes and enhances remyelination in
the cuprizone mouse model of demyelination. Neurosci Bull.; 31:
617-625; Green, A. J et al., 2017 Clemastine fumarate as a
remyelinating therapy for multiple sclerosis (ReBUILD): a
randomised, controlled, double-blind, crossover trial. Lancet Lond.
Engl. 390, 2481-2489). In our EAE model, clemastine is similarly
effective to previously published reports, but is much less
effective than nalfurafine at enabling full functional recovery
(Mei, F. et al. 2016, Accelerated remyelination during inflammatory
demyelination prevents axonal loss and improves functional
recovery. ELife 5). This example shows that nalfurafine is superior
to clemastine fumarate in this model.
Example 31: Nalfurafine Promotes a Greater and More Sustained
Recovery than Clemastine Fumarate, a Known Remyelinating Drug
Experimental detail: EAE was induced in female C57BL/6 mice as
described in Example 1. Results are shown in FIGS. 31A-1, 31A-2,
31B-1 and 31B-2. On the day of disease onset (score >1, dotted
line), mice were treated with vehicle only (daily, n=9)) or
nalfurafine (0.01 mg/kg by i.p. injection daily; n=8)(A). In a
separate similar experiment, mice were treated with vehicle only
(n=5) or clemastine fumarate (10 mg/kg by i.p. injection; n=7) (B).
Mice were considered recovered if they received a score <0.5 by
day 23 post treatment initiation. Shown are the percentage of mice
in each group that recovered (A) and the number of days in recovery
(B). ****p<0.0001 by Fisher's exact test (% recovered; A) or
one-way ANOVA with Holm-Sidak's multiple comparison test (#days in
recovery; B). These data are from the same experiments as Example
30.
Interpretation and impact: Clemastine fumarate, an anti-histamine
which also antagonizes the muscarinic receptor, has been shown to
reduce chronic disability in the EAE model when used at 10 mg/kg
starting at the time of immunization. Additionally, it has been
shown to enhance remyelination in mice and humans. In our EAE
model, clemastine fumarate treatment promotes recovery in just over
50% of the mice but the recovery is not sustained. In contrast, all
of the mice recover when treated with nalfurafine and have a
sustained recovery. This finding indicates that nalfurafine is
superior to clemastine fumarate in this model and provides a more
sustained improvement in every animal treated.
Example 32: Nalfurafine Promotes Recovery in Pain Threshold when
Administered after Demyelination in the Cuprizone Demyelination
Disease Model of MS
Experimental detail: A demyelinating disease state was induced in
female C57BL/6 mice (8-14 weeks older and between 17-23 grams in
weight). The mice were fed cuprizone-containing chow (0.3% (w/w)
cuprizone) or chow only (normal controls) for 35 days, at which
point they were switched back to standard chow. At day 28, mice
were started on daily treatment with vehicle only (DMSO: Tween 80:
Saline) or nalfurafine at 0.1 mg/kg by i.p. injection. In a second
experiment, mice were fed cuprizone-containing chow (0.3% (w/w)
cuprizone) or chow only (normal controls) for 42 days, at which
point they were switched back to standard chow. At day 35, mice
were started on daily treatment with vehicle only (DMSO: Tween 80:
Saline) or nalfurafine at 0.1 mg/kg by i.p. injection. In both
studies, on day 70, mice were culled. See FIG. 32 A for an outline
of the disease induction and treatment time course.
Sensitivity to mechanical force elicits paw withdrawal in mice.
Threshold to withdrawal is measured using calibrated von Frey
filaments using the up-down method (Bonin et al. A simplified
up-down method (SUDO) for measuring mechanical nociception in
rodents using von Frey filaments. Molecular Pain. 2014; 10:1-11) at
maximum disease, prior to treatment with nalfurafine. Cuprizone
causes increased mechanical sensitivity compared to mice on a
normal diet ({circumflex over ( )}p<0.05) (FIG. 32B), and this
increase in mechanical withdrawal threshold is reduced to baseline
levels following treatment with nalfurafine (0.1 mg/kg/i.p.).
*p<0.05 at maximum disease (day 28 or 35) and following daily
treatment with nalfurafine (average threshold days 45-70).
Nalfurafine treated mice improved mechanical threshold scores
compared to vehicle treated mice (#p<0.05). Student t-test,
n=10-11 mice/group from 2 independent experiments. {circumflex over
( )}compared to mice on normal diet; * threshold pre and post
treatment; #differences in recovery between treatment groups.
Pooled data from 2 experimental cohorts were analysed (max disease
is week prior to treatment initiation and at maximal recovery (days
63-70).
Interpretation and impact: Chronic pain is often associated with
multiple sclerosis. Allodynia is an increase in pain sensation to a
normally non-painful stimulus. In this test von Frey filaments are
used to measure the paw withdrawal threshold following application
of a defined mechanical force. Following cuprizone induced
demyelination, the pain threshold is a functional biomarker for
recovery, indicative of remyelination of the nerve fibres.
Remarkably, the diseased animals treated with nalfurafine showed a
pain sensitivity that was similar to baseline, indicating that
treatment enhances functional recovery.
* * * * *
References